CN101784669B - Improved ketoreductase polypeptides for the stereoselective production of (R)-3-hydroxythiolane - Google Patents

Improved ketoreductase polypeptides for the stereoselective production of (R)-3-hydroxythiolane Download PDF

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CN101784669B
CN101784669B CN200880104011.7A CN200880104011A CN101784669B CN 101784669 B CN101784669 B CN 101784669B CN 200880104011 A CN200880104011 A CN 200880104011A CN 101784669 B CN101784669 B CN 101784669B
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CN101784669A (en
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杰克·梁
史提芬尼·J·珍妮
艾米丽·穆德弗
拉玛·沃兰德里
詹姆士·拉伦德
吉伽特·哈思曼
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Pfizer Inc
Codexis Inc
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Pfizer Inc
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Abstract

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize chiral compounds.

Description

For the Ketoreductase polypeptides of improvement prepared by the stereoselectivity of (R)-3-hydroxy tetrahydro thiophene
1. the cross reference of related application
The application requires the rights and interests of the patent application serial numbers 60/957,974 that on August 24th, 2007 submits to according to 35 U.S.C. § 119 (e), and its content is incorporated at this by reference.
2. technical field
The disclosure relates to the purposes of engineered polypeptide and this polypeptide.
3. sequence table, the quoting of form or computer program
According to 37 C.F.R. § 1.821, through the sequence table of the computer-reader form (CRF) that EFS-Web and the application submit to simultaneously, file is called 376247-016.txt, and its entirety is incorporated to by reference.The electronic copy of sequence table results from August 24th, 2008, file size 272 Kb.
4. background
The enzyme belonging to ketoreductase (KRED) or carbonyl reduction enzyme (EC 1.1.1.184) is used for steric isomer ketone substrate synthesis of optically active alcohol before correspondence.Ketone or aldehyde substrate conversion are corresponding alcohol product by KRED usually, but all right catalysis reversed reaction, be corresponding ketone/aldehyde product by alcohol substrate oxidation.Enzyme, such as KRED, to the reduction of ketone and aldehyde with need cofactor to the oxidation of alcohol, be nicotinamide adenine dinucleotide reduced (NADH) or NADPH (NADPH) and for the Reduced nicotinamide-adenine dinucleotide (NAD) of oxidizing reaction or Triphosphopyridine nucleotide, reduced (NADP) the most commonly.NADH and NADPH is as electron donor, and NAD and NADP is as electron acceptor(EA).Often observe ketoreductase and alcoholdehydrogenase and accept phosphorylation or unphosphorylated cofactor (under its oxidation and reduced state).
KRED enzyme can find (see summary: Kraus and Waldman in large-scale bacterium and yeast, Enzyme catalysis in organic synthesis (enzyme catalysis in organic synthesis), 1st and 2 volumes, VCH Weinheim 1995; Faber, K., Biotransformations in organicchemistry (bio-transformation in organic chemistry), the 4th edition Springer, Berlin Heidelberg NewYork.2000; Hummel and Kula, 1989, Eur.J.Biochem.184:1-13).Several KRED gene and enzyme sequence are reported, such as candida magnoliae (Candida magnoliae) (Genbank accession number JC7338; GI:11360538), Candida parapsilosis (Candidaparapsilosis) (Genbank accession number BAA24528.1; GI:2815409), reddish brown shadow yeast (Sporobolomyces salmonicolor) (Genbank accession number AF 160799; GI:6539734).
In order to avoid preparing many chemosynthesis processes of key compound, the use of ketoreductase in continuous increase, so that different ketone Substrate Enzyme catalyseds is converted into chiral alcohol product.These application can adopt the full cell of expressing ketoreductase for the ketone of biocatalysis and aldehyde reduction, or in full cell, there is multiple ketoreductase wherein by those examples of the pure and mild yield of solid that adversely affects required product, adopt purifying enzyme.For external application, cofactor (NADH or NADPH) regeneration enzyme, such as glucose dehydrogenase (GDH), hydrogenlyase etc., with ketoreductase conbined usage.The example using ketoreductase to prepare useful compound comprises asymmetric reduction (Zhou, the J.Am.Chem.Soc.1983105:5925-5926 of 4-chloracetyl acetate esters; Santaniello, J.Chem.Res. (S) 1984:132-133; U.S. Patent number 5,559,030; U.S. Patent number 5,700,670 and U.S. Patent number 5,891,685); The reduction (such as U.S. Patent number 6,399,339) of dioxy carboxylic-acid; (S) reduction (such as U.S. Patent number 6,645,746 and WO01/40450) of chloro-5-hydroxyl-3-oxo hecanoic acid t-butyl ester; Based on the reduction (such as U. S. application number 2006/0286646) of the compound of pyrrolotriazine; The reduction (such as U.S. Patent number 6,800,477) of the phenyl methyl ketone replaced; And the reduction of ketone group tetramethylene sulfide (ketothiolanes) (WO 2005/054491).
Expect to differentiate to can be used for perform multiple ketone and aldehyde substrate other ketoreductases to the chiral alcohol product conversion of its correspondence.
5. summarize
The disclosure provides the Ketoreductase polypeptides of ability, the polynucleotide of this peptide species of encoding that have and 3-ketone group tetramethylene sulfide (hereinafter referred to as " substrate ") is reduced to (R)-3-hydroxy tetrahydro thiophene (hereinafter referred to as " product ") and uses the method for this polypeptide.In general, engineering Ketoreductase polypeptides of the present disclosure with available from lactobacillus kefir (Lactobacillus kefir; " L.kefir "; SEQ ID NO:4), short lactobacillus (Lactobacillus brevis; " L.brevis "; SEQ ID NO:2) and Lactobacillus minor (Lactobacillus minor; " L.minor "; SEQ ID NO:142) naturally occurring wild-type ketoreductase compare, in the chiral alcohol product that the ketone substrate conversion that will determine becomes corresponding, there is the character of improvement.The improvement of enzymic activity can comprise stereoselectivity, enzymic activity, thermostability, solvent stability, the Product inhibiton of reduction or the increase of its combination.
In some embodiments, Ketoreductase polypeptides of the present disclosure comprise wherein corresponding SEQ IDNO:2,4 or 142 the amino-acid residue of 145 of reference sequences be not acidic residues, i.e. the aminoacid sequence of L-glutamic acid or aspartic acid.So, the residue of corresponding 145 is non-acidic residue.In some embodiments, Ketoreductase polypeptides of the present disclosure has the aminoacid sequence that the residue of wherein corresponding 145 is polar residues.In some embodiments, the residue of this corresponding X145 is Serine.
In some embodiments, Ketoreductase polypeptides of the present disclosure has the reference sequences based on SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:142 (or its region or the structural domain with the residue place at corresponding X145 with Serine, such as residue 90-211) have at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, the aminoacid sequence of the identity of 99% or higher, condition is that Ketoreductase polypeptides aminoacid sequence has polar residues at the residue place of corresponding X145.In some embodiments, the residue of corresponding residue X145 is Serine.In some embodiments, Ketoreductase polypeptides based on SEQ ID NO:143,144 or 145 list type or its structural domain, such as residue 90-211, wherein the residue of corresponding X145 is polar residues, particularly Serine.In some embodiments, with reference sequences, such as SEQ IDNO:4,2 or 142 reference sequences compare, Ketoreductase polypeptides can have one or more amino acid residue difference extraly in aminoacid sequence or the structural domain determined or region.In some embodiments, the amino acid sequence differences in structural domain can comprise the combination of nonconservative aminoacid replacement, conservative aminoacid replacement and nonconservative and conservative aminoacid replacement.The multiple amino acids resi-dues that wherein can carry out this change is described in herein.
In some embodiments, substrate Stereoselective excessively with the steric isomer higher than the wild-type enzyme (i.e. SEQ ID NO:4) from lactobacillus kefir can be reduced into product by Ketoreductase polypeptides described herein.In some embodiments, substrate ketone group tetramethylene sulfide Stereoselective excessively can be reduced into product (R)-3-hydroxy tetrahydro thiophene with the steric isomer at least about 70% by Ketoreductase polypeptides described herein.
In some embodiments, the disclosure provides the High level of stereoselectivity optionally Ketoreductase polypeptides that with the steric isomer exceeding about 90% excessive (s.e.), substrate can be reduced into product.Have this High level of stereoselectivity optionally exemplary Ketoreductase polypeptides include but not limited to comprise corresponding SEQ ID NO:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106, 108, 110, 112, 126, 128, the polypeptide of the aminoacid sequence of 130 and 134.
In some embodiments, the disclosure provides the stereoselective Ketoreductase polypeptides that with the s.e. exceeding about 98%, substrate can be reduced into product.Have this stereoselective Exemplary polypeptide include but not limited to comprise corresponding SEQ ID NO:6,8,10,18,20,22,24,26,28,30,34,36,38,40,42,50,52,54,58,62,66,70,72,76,78, the polypeptide of the aminoacid sequence of 80 and 134.
In some embodiments, compared with wild-type ketoreductase, be reduced in product by the ketone determined substrate, engineering Ketoreductase polypeptides can have the enzymic activity of increase.The scope of the amount improved can nearly reach 2 times, 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times or more enzymic activitys doubly from 1.5 times of the enzymic activity of corresponding wild-type ketoreductase.In certain embodiments, engineering ketoreductase shows the enzymic activity of improvement, and its scope is 1.5 to 50 times, 1.5 to 100 times of the enzymic activity being greater than wild-type ketoreductase.Can by substrate with the Exemplary polypeptide that the speed exceeding wild-type enzyme changes into product include but not limited to comprise corresponding SEQ ID NO:6,8,10,12,14,16,18,20,22,24,26,30,32,38,40,42,44,46,48,50,52,54,56,58,60,64,66,68,70,74,76,78,80,86,88,90,92,104,106,110,112,124,126, the polypeptide of the aminoacid sequence of 130 and 134.
In some embodiments, the disclosure provides the activity and stability with the improvement exceeding wild-type enzyme, and can be greater than the Ketoreductase polypeptides that substrate to be reduced into product by about 95%s.e.The Exemplary polypeptide with this ability include but not limited to comprise corresponding SEQ ID NO:6,8,12,14,22,24,26,30,32,38,42,44,46,50,52,56,58,60,64,66,68,70,74,76,78,80,82,86,88,90,92,104,106,110, the polypeptide of the aminoacid sequence of 112 and 134.
In some embodiments, Ketoreductase polypeptides of the present disclosure, compared with wild-type, is enhanced in their thermostability, and it at high temperature contrasts according to by with wild-type, and the raising of the speed of enzymic activity determined.The exemplary Ketoreductase polypeptides with the stability of improvement include but not limited to comprise corresponding SEQ ID NO:6,8,12,14,22,24,26,32,34,36,38,42,44,46,50,52,56,58,60,64,66,68,70,74,76,78,80,82,86,88,90,92,104,106,110,112, the polypeptide of the aminoacid sequence of 124 and 134.
In yet another aspect, disclosure polynucleotide that engineering ketoreductase described herein of encoding is provided or the polynucleotide of hybridizing under high stringency with these type of polynucleotide.These polynucleotide can comprise promotor for expressing coded engineering ketoreductase and other regulatory elements, and can utilize the codon optimized for concrete required expression system.Exemplary polynucleotide includes but not limited to corresponding SEQ ID NO:5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, the nucleotide sequence of 131 and 133.
In yet another aspect, in order to operate and express the object of Ketoreductase polypeptides, the disclosure provides the host cell comprising polynucleotide and/or expression vector.These host cells can be lactobacillus kefir, short lactobacillus or Lactobacillus minor, or they can be different organisms.The expression that these host cells can be used for engineering ketoreductase described herein be separated, or alternatively, they can be directly used in 3-ketone group tetramethylene sulfide substrate conversion is chirality (R)-3-hydroxy tetrahydro thiophene product.
No matter perform described method with the ketoreductase of full cell, cell extract or purifying, single ketoreductase can be used, or alternatively, the mixture of two or more ketoreductases can be used.
As implied above, ketoreductase described herein can ketone group in the compound 3-ketone group tetramethylene sulfide of catalytic structure formula (I)
To the reduction reaction of chiral alcohol product (the R)-3-hydroxy tetrahydro thiophene of counter structure formula (II)
Therefore, in some embodiments, the disclosure provides method 3-ketone group tetramethylene sulfide (" substrate ") being reduced into (R)-3-hydroxy tetrahydro thiophene (" product "), described method comprise with Ketoreductase polypeptides of the present disclosure be suitable for by substrate reduction or be converted into product reaction conditions under contact or hatch substrate.In some embodiments of this method, substrate is reduced into have and at least exceedes about 65% or at least exceed the excessive product of the steric isomer of wild-type.
In some embodiments of this method, substrate is reduced into product to exceed about 90%s.e., wherein said Ketoreductase polypeptides comprise corresponding SEQ ID NO:6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,86,88,90,92,94,96,100,102,104,106,108,110,112,126,128, the aminoacid sequence of 130 and 134.
In some embodiments of this method, substrate is reduced into product to exceed about 98%s.e., wherein said Ketoreductase polypeptides comprise corresponding SEQ ID NO:6,8,10,18,20,22,24,26,28,30,34,36,38,40,42,50,52,54,58,62,66,70,72,76,78, the aminoacid sequence of 80 and 134.
At this for substrate being reduced in some embodiments of the method for product, contrast with wild-type enzyme (SEQ ID NO:4), substrate is with at least 1.5 times, 2 times, 3 times 4 times, 5 times, 10 times, the speed of 20 times or more raisings is doubly reduced into product, wherein said Ketoreductase polypeptides comprises corresponding SEQ ID NO:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 30, 32, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 64, 66, 68, 70, 74, 76, 78, 80, 86, 88, 90, 92, 104, 106, 110, 112, 124, 126, the aminoacid sequence of 130 and 134.
In some embodiments, described method relates to Ketoreductase polypeptides at other compounds of synthesis, the purposes in such as medical compounds.In some embodiments, described method relates to Ketoreductase polypeptides and has purposes in the microbiotic sulopenem (CP-70,429) of following structural formula (III) in synthesis:
Therefore, in some embodiments, at microbiotic (the i.e. CP-70 of composite structure formula (III), 429) in method, step in method can be included in be suitable for by the substrate conversion of structural formula (I) or be reduced into structural formula (II) the reaction conditions of product under, with the substrate of any ketoreductase contact (I) described herein.The compound of the structural formula (II) that the steric isomer of at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% or higher is excessive can be used for the compound of preparation formula (III).
6. accompanying drawing summary
Fig. 1 explains the effect of ketoreductase (KRED) in chiral alcohol product (the R)-3-hydroxy tetrahydro thiophene compound 3-ketone group tetramethylene sulfide of substrate formula (I) being changed into corresponding formula (II).In this reaction, substrate is reduced into corresponding (R)-ol by biocatalysis.This reduction uses the cofactor of KRED of the present disclosure and such as NADPH.Glucose dehydrogenase (GDH) can be used for NADP +change/recirculate to NADPH.Conversion of glucose becomes glyconic acid, and it transfers to change into its sodium salt (gluconic acid sodium salt) by adding sodium hydroxide.Embodiment 9 provides the non-limiting method for carrying out this reaction.
7. describe in detail
7.1 definition
As used herein, following term is intended to have following implication.
" ketoreductase" and " kRED" mutually can exchange the polypeptide used to refer to have carbonyl reduction the enzymatic ability being the alcohol of its correspondence in this article.More specifically, the compound Stereoselective of above-mentioned formula (I) can be reduced to the product of corresponding above-mentioned formula (II) by Ketoreductase polypeptides described herein.This polypeptide utilizes cofactor nicotinamide adenine dinucleotide reduced (NADH) or NADPH (NADPH) as reductive agent usually.As used herein, ketoreductase comprises the engineered polypeptide that naturally occurring (wild-type) ketoreductase and the non-natural that produced by manual handling exist.
" encoding sequence" refer to the part nucleic acid (such as gene) of the aminoacid sequence of coded protein.
" naturally occurring" or " wild-type" refer to occurring in nature find form.Such as, naturally occurring or wild type peptide or polynucleotide sequence are the sequences being present in organism, and it can be separated from the source of occurring in nature, and is not expressly modified by manual handling.
When use relates to such as, when cell, nucleic acid or polypeptide, " restructuring" refer to what the mode do not existed in addition with occurring in nature was modified, or identical with it, but be by synthetic materials and/or by using the process of recombinant technology to prepare or derivative material, or correspond to the material of natural or natural formation of material.Wherein, nonrestrictive example comprises the reconstitution cell of expressing the intrinsic gene that undiscovered gene or expression are expressed in addition with different levels in intrinsic (non-recombinant) form of cell.
" the per-cent of sequence iden" and " percent homology" be used interchangeably the contrast that refers between polynucleotide and polypeptide in this article; and to determine by contrasting two best aligned sequences in contrast form (comparison window); the polynucleotide wherein in contrast form or peptide sequence part contrast with the reference sequences (its do not comprise adds or lack) for the best comparison of two sequences, can comprise interpolation or lack (i.e. breach).This per-cent is by following calculating: determine that positional number that identical nucleic acid base or amino-acid residue all exist two sequences is to produce matched position number, by the sum of matched position number divided by position in contrast form, and be multiplied by result to produce the per-cent of sequence iden with 100.Alternatively, this per-cent is by following calculating: determine identical nucleic acid base or amino-acid residue two sequences all exist or the positional number of nucleic acid base or amino-acid residue and breach comparison to produce matched position number, this matched position number divided by the total number of positions in contrast form, and is multiplied by result to produce the per-cent of sequence iden with 100.It will be understood by those skilled in the art that many algorithms that can be used for the determination of comparison two sequences.Best comparison for the sequence contrasted is undertaken by following: the local homology algorithm of such as Smith and Waterman, 1981, Adv.Appl.Math.2:482, the homology alignment algorithm of Needleman and Wunsch, 1970, J.Mol.Biol.48:443, the search for similarity method of Pearson and Lipman, 1988, Proc.Natl.Acad.Sci.USA85:2444, (the GAP in GCG Wisconsin software package is performed by the computerize of these algorithms, BESTFIT, FASTA and TFASTA), or range estimation is (generally see Current Protocols inMolecular Biology (up-to-date experimental methods of molecular biology compilation), the people such as F.M.Ausubel compile, Current Protocols, Greene Publishing Associates, Inc. with John Wiley & Sons, Inc. cooperate between, (1995 supplement) (Ausubel)).The example being suitable for the algorithm determining Percent sequence identity and sequence similarity is BLAST and BLAST 2.0 algorithm, they are described in the people such as Altschul respectively, the people such as 1990, J.Mol.Biol.215:403-410 and Altschul, 1977, Nucleic Acids Res.3389-3402.The software analyzed for performing BLAST is publicly available by the website at American National Biotechnology Information center.This algorithm comprises first by differentiating that the short word of length W differentiates high score sequence pair (HSP) in search sequence, mates or meets some on the occasion of threshold score T during the word comparison of described short word with equal length in database sequence.T is called neighborhood word score threshold (people such as Altschul, the same).These initial neighbourhood word coupling is as starting the seed of search to find the longer HSP containing them.Then, the both direction of word coupling along each sequence is extended, until accumulation comparison score can be increased.For nucleotide sequence, operation parameter M (the award score that coupling residue is right; Always > 0) and the N (point penalty of mismatched residue; Always < 0) calculate accumulation score.For aminoacid sequence, score matrix is for calculating accumulation score.When following situation, the extension of word in each direction coupling is terminated: accumulation comparison score maximumly reaches the value amount of have dropped X from it; Owing to accumulating one or more negative scoring residue alignments, accumulation score reaches 0 or following; Or arrive arbitrary sequence end.BLAST algorithm parameter W, T and X determine susceptibility and the speed of comparison.BLASTN program (for nucleotide sequence) uses word length (W) 11, expected value (E) 10, the contrast of M=5, N=-4 and two chains is worth by default.For aminoacid sequence, BLASTP program uses word length (W) 3, expected value (E) 10 and BLOSUM62 score matrix to be worth (see Henikoff and Henikoff by default, 1989, Proc Natl AcadSci USA 89:10915).Sequence alignment and the exemplary of % sequence iden are determined to use BESTFIT or the GAP program in GCG Wisconsin software package (Accelrys, Madison WI), use the default parameters provided.
" reference sequences" refer to for the fixed sequence really of the basis as alignment.Reference sequences can be the section of the subset of larger sequence, such as full-length gene or peptide sequence.In general, reference sequences is the total length of at least 20 Nucleotide or amino acid residues length, at least 25 residues in length, at least 50 residues in length or nucleic acid or polypeptide.Because two polynucleotide or polypeptide (1) may be included in sequence (i.e. a part for complete sequence) similar between two sequences separately, and (2) sequences different between two sequences can be comprised further, the alignment between therefore two (or more) polynucleotide or polypeptide usually by the sequence of contrast " contrast form " upper two polynucleotide to differentiate that the regional area of also comparative sequences similarity carries out.
In some embodiments, " reference sequences " can based on primary amino acid sequences, and wherein reference sequences is the sequence can in primary sequence with one or more change.Such as, reference sequences refers to that the residue that the X145 of wherein SEQ IDNO:4 is corresponding changes into Serine " to have the SEQ ID NO:4's of Serine based on the residue place at corresponding X145 ".
" contrast form" refer at least about 20 continuous nucleotide positions or amino-acid residue notional section; wherein sequence can contrast with at least 20 continuous nucleotides or amino acid whose reference sequences; and wherein the Sequence contrasted in form can comprise compared with the reference sequences of the best comparison for two sequences (its do not comprise add or lack), and 20% or less interpolation or disappearance (i.e. breach).Contrast form can be longer than 20 continuous print residues, and comprise optionally 30,40,50,100 or longer form.
" basic identity" to refer in the contrast form of at least 20 resi-dues, usually contrast with reference sequences in the form of at least 30-50 residue; have the sequence iden of at least 80%, the identity of at least 85% and 89% to 95% sequence iden; more commonly at least 99% the polynucleotide of sequence iden or peptide sequence, wherein the per-cent of sequence iden be by contrast form, contrast reference sequences and comprise add up to 20% of reference sequences or the sequence of less disappearance or interpolation calculate.In the specific embodiments being applied to polypeptide, term " basic identity " refers to ought such as by program GAP or BESTFIT, when using default gap weight to carry out best comparison, two peptide sequences share the sequence iden (sequence iden of such as 99%) of sequence iden, the preferably sequence iden of at least 89%, the sequence iden of at least 95% or more of at least 80%.Preferably, different amino acid residue positions is different because conserved amino acid replaces.
When using when the numbering of given amino acid or polynucleotide sequence, " corresponding in", " reference" or " relative to" refer to when given amino acid or polynucleotide sequence and the reference sequences of specifying contrast, the numbering of the residue of described reference sequences.In other words, given polymeric residue numbering or resi-dues are specified according to reference sequences, instead of are specified by the actual numbered positions of residue in given amino acid or polynucleotide sequence.Such as, given aminoacid sequence, the such as aminoacid sequence of engineering ketoreductase, by importing breach and reference sequences comparison to optimize the residue match between two sequences.In these cases, although there is breach, the residue in given amino acid or polynucleotide sequence is numbered according to the reference sequences of its comparison.
" stereoselectivity" refer to the preferential formation of a steric isomer compared with another steric isomer in chemistry or enzymatic reaction.Stereoselectivity can be part, and the formation of one of them steric isomer is favourable relative to another steric isomer, or it can be completely, wherein only forms a steric isomer.When steric isomer is enantiomorph, stereoselectivity is called enantioselectivity, the ratio of an enantiomorph in two summations (being usually reported as per-cent).It generally reports that (being generally per-cent) is the enantiomeric excess (e.e) therefrom calculated according to formula [major enantiomer-secondary enantiomorph]/[major enantiomer+secondary enantiomorph] in the art alternatively.This also can be called steric isomer excessive (s.e).If steric isomer is diastereomer, then stereoselectivity is called cis-selectivity, the ratio (be usually reported as per-cent) of a diastereomer in the mixture of two non-corresponding.
" high level of stereoselectivity selectivity" referring to can be excessive in substrate conversion or the Ketoreductase polypeptides being reduced to corresponding (R)-product with the steric isomer at least about 85%.
" the enzymatic property improved" refer to and the Ketoreductase polypeptides showing improvement compared with ketoreductase in any enzymatic property.For engineering Ketoreductase polypeptides described herein, usual and wild-type ketoreductase contrasts, although in some embodiments, can be the engineering ketoreductase that another improves with reference to ketoreductase.The enzymatic property improved is needed to include but not limited to, enzymatic activity (it can be expressed according to the conversion percentages of substrate), thermostability, pH living features, cofactor requirements, refractoriness (such as Product inhibiton), stereospecificity and stereoselectivity (comprising enantioselectivity) to inhibitor.
" the enzymatic activity increased" refer to the character of the improvement of engineering Ketoreductase polypeptides; it can be expressed as and contrast with reference to ketoreductase; specific activity (such as produced product/time/weight albumen) increase; or substrate conversion is the increase of the per-cent (when such as using the KRED of specified amount, the substrate of initial amount is converted into the per-cent of product within the time period of specifying) of product.The illustrative methods determining enzymic activity is provided in an embodiment.Any character relating to enzymic activity may be affected, comprise typical enzymatic property K m, V maxor k cat, their change can cause the increase of enzymatic activity.The improvement of enzymic activity can be about 1.5 times of the enzymatic activity from corresponding wild-type ketoreductase, and the enzymatic activity to the naturally occurring ketoreductase or another engineering ketoreductase that exceed derivative described Ketoreductase polypeptides reaches 2 times, 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times or more.In particular embodiments, engineering ketoreductase can show the enzymatic activity of improvement, and its scope is larger than the enzymatic activity of parent ketoreductase 1.5 to 50 times or 1.5 to 100 times.It will be understood by those skilled in the art that the activity of any enzyme is diffusional limitation, so that catalysis turn-around speed can not exceed the rate of diffusion of substrate (comprising any required cofactor).The theoretical maximum of diffusional limitation, or k cat/ K m, be generally about 10 8to 10 9(M -1s -1).Therefore, any improvement of the enzymic activity of ketoreductase is by the upper limit relevant for the rate of diffusion with the substrate acted on ketoreductase.Ketoreductase is active to be measured by any one measurement in ketoreductase standard test used, the NADPH such as measured owing to being reduced into alcohol with ketone or aldehyde is oxidized the reduction (see embodiment 6) of NADPH absorbancy or the fluorescence caused, or is measured by the product produced in coupling mensuration.As described in further detail herein, use determine enzyme preparation, under imposing a condition, location survey is fixed really and one or more substrate determined carries out the contrast of enzymic activity.In general, when contrasting lysate, the quantity determining cell and the amount of protein measured, and use identical expression system and identical host cell, minimize with the change of the amount making enzyme that is that host cell produces and that exist in lysate.
" transform" refer to Substrate Enzyme catalysed to be reduced to corresponding product." conversion percentages " refers to the per-cent of the substrate being reduced to product under the specified conditions in certain hour section.Therefore, " enzymatic activity " or " activity " of Ketoreductase polypeptides can be expressed as " conversion percentages " of substrate to product.
" thermally-stabilised" refer to, after the temperature being exposed to rising (such as 40-80 DEG C) for some time (such as 0.5-24 hour), contrast with untreated enzyme, maintain the Ketoreductase polypeptides of similar activity (such as more than 60% to 80%).
" solvent-stable" refer at the solvent being exposed to varied concentration (such as 5-99%) (such as Virahol, tetrahydrofuran (THF), 2-methyltetrahydrofuran, acetone, toluene, N-BUTYL ACETATE, methyl tertiary butyl ether etc.) after for some time (such as 0.5-24 hour); contrast with untreated enzyme, maintain the Ketoreductase polypeptides of similar activity (exceeding such as 60% to 80%).
" pH stablizes" refer to, after being exposed to high or low pH (such as 4.5-6 or 8 to 12) for some time (such as 0.5-24 hour), contrast with untreated enzyme, maintain the Ketoreductase polypeptides of similar activity (exceeding such as 60% to 80%).
" thermally-stabilised and solvent-stable" refer to the not only thermally-stabilised but also Ketoreductase polypeptides of solvent-stable.
As used when engineering ketoreductase herein, " derived from" differentiate the gene of the starting ketone reductase enzyme that relies on of engineering and/or this type of ketoreductase of encoding.Such as, the engineering ketoreductase of SEQ ID NO:134 is obtained by many for the encode gene of lactobacillus kefir ketoreductase of SEQ ID NO:4 of artificial evolution.Therefore, this engineering ketoreductase " derived from " the wild-type ketoreductase of SEQ ID NO:4.
" hydrophilic amino acid or residue" refer to have and show according to people such as Eisenberg, hydrophobicity scale is unified in the stdn of 1984, J.Mol.Biol.179:125-142, the amino acid of the minus side chain of hydrophobicity or residue.The hydrophilic amino acid of genetic coding comprises L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K) and L-Arg (R).
" acidic amino acid or residue" refer to when amino acid is contained in peptide or polypeptide, there is hydrophilic amino acid or the residue of the side chain showing the pK value being less than about 6.At physiological ph, acidic amino acid has electronegative side chain usually owing to having lacked hydrogen ion.The acidic amino acid of genetic coding comprises L-Glu (E) and L-Asp (D).
" basic aminoacids or residue" refer to when amino acid is contained in peptide or polypeptide, there is hydrophilic amino acid or the residue of the side chain showing the pK value being greater than about 6.At physiological ph, basic aminoacids has the side chain of positively charged usually owing to associating with oxonium ion.The basic aminoacids of genetic coding comprises L-Arg (R) and L-Lys (K).
" polare Aminosaeren or residue" refer to that there is physiological pH under the hydrophilic amino acid of uncharged side chain or residue, but this side chain has the electron pair that wherein two atoms share is maintained more close at least one key of one of described atom.The polare Aminosaeren of genetic coding comprises L-Asn (N), L-Gln (Q), L-Ser (S) and L-Thr (T).
" hydrophobic amino acid or residue" refer to have and show according to people such as Eisenberg, hydrophobicity scale is unified in the stdn of 1984, J.Mol.Biol.179:125-142, and hydrophobicity is greater than amino acid or the residue of the side chain of zero.The hydrophobic amino acid of genetic coding comprises L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y).
" aromatic amino acid or residue" refer to have the hydrophilic of the side chain comprising at least one aromatic ring or heteroaromatic rings or hydrophobic amino acid or residue.The aromatic amino acid of genetic coding comprises L-Phe (F), L-Tyr (Y) and L-Trp (W).Although the pKa of the heteroaromatic nitrogen-atoms due to L-His (H), it is classified as alkaline residue sometimes, or be categorized as aromatic moieties because its side chain comprises heteroaromatic rings, but Histidine is classified as hydrophilic residue or is " restriction residue " (seeing below) herein.
" limiting amino acid or residue" refer to that there is the geometric amino acid of restriction or residue.Herein, limit residue and comprise L-pro (P) and L-his (H).Histidine has restriction geometry, is because it has relatively little imidazole ring.Proline(Pro) has restriction geometry, is also because it also has five-ring.
" nonpolar amino acid or residue" refer to that there is physiological pH under the hydrophobic amino acid of uncharged side chain or residue, and this side chain has the key (namely side chain is nonpolar) that electron pair that wherein two atoms share generally is maintained by each equality in two atoms.The nonpolar amino acid of genetic coding comprises L-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M) and L-Ala (A).
" aliphatic amino acid or residue" refer to hydrophobic amino acid or the residue with aliphatic hydrocarbon side chain.The aliphatic amino acid of genetic coding comprises L-Ala (A), L-Val (V), L-Leu (L) and L-Ile (I).
" halfcystine".Amino acid L-Cys (C) is uncommon, and reason is that the amino acid that it can contain sulfanyl or sulfhedryl with other L-Cys (C) amino acid or other forms disulphide bridges." cysteine-like residue " comprises halfcystine and containing other amino acid of sulfhydryl moiety that can be used for being formed disulphide bridges.Whether the capacity L-Cys (C) that L-Cys (C) (with other amino acid of the side chain had containing-SH) is present in peptide with the disulphide bridges form of dissociate-SH or the oxidation of reducing contributes hydrophobic or hydrophilic profile only to peptide.Although according to the stdn uniform scale of Eisenberg (people such as Eisenberg, 1984, the same), L-Cys (C) shows as the hydrophobicity of 0.29, but should be understood that in order to object of the present disclosure, L-Cys (C) himself is divided into separately one group.
" little amino acid or residue" refer to have amino acid or the residue of the side chain formed primarily of total three or less carbon and/or heteroatoms (getting rid of alpha-carbon and hydrogen).According to above definition, little amino acid or residue can be categorized as little amino acid or the residue of aliphatics, nonpolar, polarity or acidity further.The p1 amino acid of genetic coding comprises L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and L-Asp (D).
" amino acid containing hydroxyl or residue" refer to containing hydroxyl (-OH) amino acid partly.The amino acid containing hydroxyl of genetic coding comprises L-Ser (S), L-Thr (T) and L-Tyr (Y).
" conservative" aminoacid replacement or sudden change refer to the interchangeability of the residue with similar side chain therefore generally include the amino acid with belonging in the aminoacid replacement polypeptide of same or analogous amino acid define styles.But, as used herein, in some embodiments, if conservative variants can be alternatively from aliphatic residue to aliphatic residue, from non-polar residue to non-polar residue, from polar residues to polar residues, from acidic residues to acidic residues, from alkaline residue to alkaline residue, from aromatic moieties to aromatic moieties or limit the replacement of residue to restriction residue, conservative variants does not comprise from hydrophilic residue to hydrophilic residue, from hydrophobic residue to hydrophobic residue, from the residue containing hydroxyl to the residue containing hydroxyl or from little residue to the replacement of little residue.In addition, as used herein, A, V, L or I conservatively can be sported another aliphatic residue or another non-polar residue.Following table display Exemplary conservative replace.
Residue Possible conservative variants
A、L、V、I Other non-polar residue (A, L, V, I, G, M) of other aliphatic residue (A, L, V, I)
G、M Other non-polar residue (A, L, V, I, G, M)
D、E Other acidic residues (D, E)
K、R Other alkaline residue (K, R)
P、H Other restriction residue (P, H)
N、Q、S、T Other polar residues
Y、W、F Other aromatic moieties (Y, W, F)
C Nothing
" non-conservative substitutions" refer to the amino acid in the significantly different aminoacid replacement of side chain properties or mutant polypeptide.Non-conservative substitutions can use the amino acid between definitions section listed above, instead of belongs to the amino acid of one group.In one embodiment, non-conservative sudden change impact (a) replaces structure (such as proline for glycine) (b) electric charge or the hydrophobicity of peptide main chain in region, or the volume of (c) side chain.
" disappearance" refer to that the one or more amino acid by removing reference polypeptide carry out modified polypeptide.Disappearance in aminoacid sequence can comprise 1 of removal composition reference enzyme an or more amino acid, 2 or more amino acid, 3 or more amino acid, 4 or more amino acid, 5 or more amino acid, 6 or more amino acid, 8 or more amino acid, 10 or more amino acid, 15 or more amino acid, or 20 or more amino acid, reach 10% of amino acid sum, reach 15% of amino acid sum or reach 20% of amino acid sum, and retain the character of the improvement of enzymatic activity and/or reservation engineering ketoreductase.Disappearance can for the internal portion of polypeptide and/or terminal portions.In different embodiments, it can be maybe discontinuous that disappearance can comprise continuous print section.
" insert" refer to compared with reference polypeptide, carry out modified polypeptide by adding one or more amino acid.In some embodiments, the engineering ketoreductase of improvement comprises and inserts one or more amino acid to naturally occurring Ketoreductase polypeptides, and inserts one or more amino acid to the Ketoreductase polypeptides of other improvement.Can insert at the internal portion of polypeptide, or be inserted into carboxyl or N-terminal.As used herein, insert and comprise fusion rotein known in the art.Insertion can be continuous print stretches of amino acids, or is separated by the one or more amino acid in naturally occurring polypeptide.
" fragment" refer to that there is N-terminal and/or carboxyl-terminal deletion, but the polypeptide that wherein remaining aminoacid sequence is identical with the correspondence position in sequence.Fragment can be at least 14 amino acid longs, at least 20 amino acid longs, at least 50 amino acid longs or longer, and reaches 70%, 80%, 90%, 95%, 98% and 99% of total length Ketoreductase polypeptides.In some embodiments, described total length ketoreductase can be total length engineering or wild-type Ketoreductase polypeptides (such as SEQ ID NO:2 and 4).
" isolated polypeptide" refer to from other impurity (as protein, lipid and the polynucleotide) isolated polypeptide substantially with natural adjoint polypeptide.This term comprises and shifting out from the naturally occurring environment or expression system (such as host cell or external synthesis) of polypeptide or the polypeptide of purifying.The ketoreductase improved can be present in cell, be present in cell culture medium, or prepared by the goods of such as lysate or separation in different forms.Therefore, in some embodiments, the ketoreductase of improvement can be isolated polypeptide.
" substantially pure polypeptide" refer to a kind of composition; wherein polypeptide thing class is the dominant class (i.e. basis mole or weight; it is abundanter than the single macromolecular complex of any other in composition class) existed; and when target compound class with mole or the % weighing scale macromolecular complex class that comprises existence is at least about 50% time, said composition is generally the composition of basic purifying.In general, substantially pure ketoreductase composition by mole or % weighing scale, will be present in all macromolecular complex classes in composition about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more and about 98% or more be comprised.In some embodiments, be basic homogeneity (namely can not impurity species be detected by common detection methods in the composition) by target compound class purifying, wherein said composition is made up of single macromolecular complex class substantially.Solvent thing class, small molecules (< 500 dalton) and element ion thing class do not think macromolecular complex class.In some embodiments, the Ketoreductase polypeptides of the improvement of separation is substantially pure peptide composition.
Used herein " stingent hybridization" refer to the condition that nucleic acid hybrids is stable.As is known to the person skilled in the art, the stability of crossbred reflects with the melting temperature(Tm) of crossbred (Tm).In general, the stability of crossbred is the function of the existence of ionic strength, temperature, G/C content and chaotropic agent.The Tm value of polynucleotide can use the currently known methods for predicting melting temperature(Tm) to calculate (see people 1989, MethodsEnzymology 168:761-777 such as such as Baldino; The people such as Bolton, 1962, Proc.Natl.Acad.Sci.USA 48:1390; The people such as Bresslauer, 1986, Proc.Natl.Acad.Sci USA 83:8893-8897; The people such as Freier, 1986, Proc.Natl.Acad.Sci USA83:9373-9377; The people such as Kierzek, Biochemistry 25:7840-7846; The people such as Rychlik, 1990, Nucleic Acids Res 18:6409-6412 (corrigenda, 1991, Nucleic Acids Res19:698); The people such as Sambrook, the same; The people such as Suggs, 1981, in the Developmental BiologyUsing Purified Genes developmental biology of purified genes (use) people such as (compile) Brown, 683-693 page, Academic Press; And Wetmur, 1991, Crit Rev Biochem Mol Biol26:227-259; All publications are incorporated to by reference at this).In some embodiments, polynucleotide encoding polypeptide disclosed herein, and determining to hybridize under condition (such as appropriateness strict or high stringency) complementary sequence of sequence of engineering ketoreductase disclosed in the code book.
" hybridization stringency" refer in the hybridization of nucleic acid, the hybridization conditions of such as wash conditions.In general, hybridization carries out under the condition of lower severity, washs subsequently under the still higher severity of change.Term " appropriate stingent hybridization " refers to the condition allowing target dna in conjunction with complementary nucleic acid, described complementary nucleic acid and target dna have identity, the preferably identity of about 75%, the identity of about 85% of about 60%, and itself and herbicide-tolerant polynucleotide have the identity being greater than about 90%.Exemplary appropriate stringent condition is equal to hybridize in 50% methane amide, 5 × Denhart solution, 5 × SSPE, 0.2%SDS at 42 DEG C, the condition of washing in 0.2 × SSPE, 0.2%SDS at 42 DEG C subsequently." high stringency hybridization" refer generally to as determined under the solution condition of the polynucleotide sequence determined, than thermal melting point T mlow about 10 DEG C or less condition.In some embodiments, high stringency refers to only allow at 65 DEG C, form the condition stablizing those nucleic acid array hybridizings of crossbred in 0.018M NaCl.(namely as herein institute is contained, if crossbred instability in 0.018M NaCl at 65 DEG C, so it is also unstable under high stringency).Such as, provide high stringency by following: hybridize under the condition being equal to 50% methane amide at 42 DEG C, 5 × Denhart solution, 5 × SSPE, 0.2%SDS, wash in 0.1 × SSPE and 0.1%SDS at 65 DEG C subsequently.Another high stringency is hybridized under the condition of 5 × SSC containing 0.1% (w:v) SDS being equal at 65 DEG C, and washs in containing the 0.1 × SSC of 0.1%SDS at 65 DEG C.Other high stringency conditions, and appropriate stringent condition describes in reference cited above.
" allos" polynucleotide refer to any polynucleotide of room technological sourcing host cell by experiment, and comprise and shifting out from host cell, carry out laboratory treatment, and then import the polynucleotide of host cell.
" the codon optimized" refer to that the codon of the polynucleotide of coded protein is changed to the preferential codon used in specific organism, so that coded protein is effectively expressed in the organism paid close attention to.Although genetic code is degeneracy, namely by several, most of amino acid is called that the codon of " synonym " or " synonym " codon represents, and the use of known specific organism to codon is nonrandom and prefers to specific codon triplet.For the protein that the gene of given gene, common function or ancestors' origin, the Protein high of relative low copy number are expressed, and the genomic collectin matter coding region of organism, this codon usage bias may be higher.In some embodiments, encode ketoreductase polynucleotide may in order to select to have carried out for best production in the host organisms of expressing codon optimized.
" preferably, best, high codon uses preferred codons" refer to codon higher than other codon usage frequencies of coding same amino acid in protein coding region interchangeably.The determination of preferred codon can with following about: the codon in individual gene, one group of gene with common function or origin, the gene of highly expressing uses; Codon frequency in the collectin matter coding region of whole organism; Codon frequency in the collectin matter coding region of related organisms or its combination.The best codon of codon normally for expressing that frequency increases with gene expression dose.Become known for the multiple method determining codon frequency in concrete organism (such as codon use, Relative synomons Codon use) and codon hobby, comprise multivariate analysis, such as use cluster analysis or correspondence analysis, and in gene the significant figure of codon used (see GCG CodonPreference, GeneticsComputer Group Wisconsin Package; CodonW, John Peden, University ofNottingham; McInerney, J.O, 1998, Bioinformatics 14:372-73; The people such as Stenico, 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29).Can obtain ever-increasing organism list codon use table (see people such as such as Wada, 1992, NucleicAcids Res.20:2111-2118; The people such as Nakamura, 2000, Nucl.AcidsRes.28:292; The people such as Duret, the same; Henaut and Danchin, " Escherichia coli andSalmonella (intestinal bacteria and Salmonellas) ", the people such as 1996, Neidhardt compile, ASM Press, Washington D.C., 2047-2066 page).The data source used for obtaining codon can be dependent on can any available core nucleotide sequence of coded protein.These data sets comprise the nucleotide sequence in the predictive coding district of protein (such as complete protein encoding sequence-CDS), the sequence label (ESTS) of expressing or the genome sequence that actual known coded is expressed (see such as Mount, D., Bioinformatics:Sequence and Genome Analysis (information biology: sequence and genome analysis), 8th chapter, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y, 2001; Uberbacher, E.C., 1996, Methods Enzymol.266:259-281; The people such as Tiwari, 1997, Comput.Appl.Biosci.13:263-270).
Defined herein " control sequence" comprise the polynucleotide paid close attention to or the expression of polypeptide is required or favourable all components.For the nucleotide sequence of coded polypeptide, each control sequence can be intrinsic or external source.This control sequence includes but not limited to, leader sequence, polyadenylation se-quence, propeptide sequence, promotor, signal peptide sequence and transcription terminator.At least, control sequence comprises promotor, and transcribes and translation termination signal.The control sequence with linker can be provided, to import the concrete restriction site promoting that described control sequence is connected with the coding region of the nucleotide sequence of coded polypeptide.
" be operably connected" be defined as a kind of configuration in this article; wherein control sequence is suitably to place with functional relationship (being namely in relative position) with the polynucleotide paid close attention to or polypeptide (encoding sequence in such as DNA sequence dna), so that control sequence instructs or regulates the expression of polynucleotide and/polypeptide.
" promoter sequence" be the nucleotide sequence identified by the host cell of the polynucleotide (such as coding region) for expressing concern.In general, promoter sequence comprises the transcriptional control sequence of the expression of the polynucleotide that mediation is paid close attention to.Promotor can be show any nucleotide sequence of transcriptional activity in the host cell selected, and comprises sudden change, brachymemma and hybrid promoter, and can from the homology of host cell or the Codocyte of allos or the gene of intracellular polypeptides obtain.
" cofactor regeneration system" refer to reaction (the such as NADP of the cofactor participating in reduction-oxidation form +to NADPH) a group reaction thing.The cofactor be oxidized by the reduction of the enzymatic ketone substrate of ketone body powder is regenerated as reduction form by cofactor regeneration system.Cofactor regeneration system comprises stoichiometric reductive agent, and it is the source going back protohydrogen equivalent, and can the cofactor of reduction-oxidation form.Cofactor regeneration system can comprise catalyzer further, such as enzyme catalyst, the cofactor of its catalytic reducer reduction-oxidation form.From NAD +or NADP +the cofactor regeneration system of regeneration of NAD H or NADPH is known in the art respectively, and can be used in method described herein.
7.2 ketoreductase
The disclosure provides engineering ketoreductase (" KRED "), and it can by the 3-ketone group tetramethylene sulfide (" substrate ") of structural formula (I)
Stereoselective reduction or be converted into (R)-3-hydroxy tetrahydro thiophene (" product ") of structural formula (II):
And when contrasting with the naturally occurring wild-type KRED enzyme available from lactobacillus kefir (SEQ ID NO:4), short lactobacillus (SEQ ID NO:2) or Lactobacillus minor (SEQ ID NO:142), or when contrasting with other engineering ketoreductase, there is the character of improvement.The enzymatic property improved is needed to include but not limited to enzymatic activity, thermostability, pH living features, cofactor requirements, refractoriness (such as Product inhibiton), stereospecificity, stereoselectivity and solvent stability to inhibitor.Improvement can relate to the combination of single enzymatic property (such as enzymatic activity) or different enzymatic property (such as enzymatic activity and stereoselectivity).
The disclosure provides the engineering ketoreductase (" KRED ") that the ketone substrate Stereoselective determined can be reduced to the alcohol product of its correspondence, and this enzyme is when contrasting with the naturally occurring wild-type KRED enzyme available from lactobacillus kefir (SEQ IDNO:2) or short lactobacillus (SEQ ID NO:4) or Lactobacillus minor (SEQ ID NO:142), or when contrasting with Other Engineering ketoreductase, there is the character of improvement.The residue that Ketoreductase polypeptides of the present disclosure has 145 of corresponding SEQ ID NO:2 or 4 or 142 is not the demand of acidic residues.In some embodiments, with the lactobacillus kefir of SEQ ID NO:2 or 4 or 142 or short lactobacillus or Lactobacillus minor KRED alignment, Ketoreductase polypeptides has polare Aminosaeren at the residue place of corresponding 145.In some embodiments, with the lactobacillus kefir of SEQID NO:2 or 4 or 142 or short lactobacillus or Lactobacillus minor KRED alignment, Ketoreductase polypeptides has Serine at the residue place of corresponding 145.
As implied above, in some embodiments, with reference to the lactobacillus kefir ketoreductase of SEQ ID NO:4, the short lactobacillus ketoreductase of SEQ ID NO:2, the Lactobacillus minor of SEQ ID NO:142 or engineering ketoreductase, the engineering ketoreductase with the enzymic activity of improvement is described.In these ketoreductases, amino acid residue position is determined (namely M represents 1 residue) from initial methionine(Met) (M) residue, although it will be appreciated by those skilled in the art that, such as in host cell or external translating system, this initial methionine residues removes to produce the mature protein lacking initial methionine residues by biological processing system.The amino acid residue position of the specific amino acids existed in aminoacid sequence or amino acid change is described as term " Xn " or " n position " in this article sometimes, and wherein n refers to resi-dues.If the amino-acid residue between ketoreductase on identical residue position is different, then different residues is arranged as such as " lactobacillus kefir residue/short lactobacillus residue/Lactobacillus minor " by "/" use and indicates.In some embodiments, replace sudden change, it is the displacement replacing the amino-acid residue in the corresponding residue of reference sequences (such as the wild-type ketoreductase of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:142) with different amino-acid residues, indicates with symbol " → ".
Herein, sudden change is described as the amino acid of sudden change " one-tenth is a kind of " type sometimes.Such as, residue X7 can by glycine mutation " one-tenth is a kind of " serine residue.But another amino acid becoming identical type from an amino acid mutation of a type is not got rid of in the use that phrase " becomes a kind of ".Such as, residue X7 can become arginine from glycine mutation.
Encoding naturally occurring lactobacillus kefir, the polynucleotide sequence of ketoreductase (being also called " ADH " or " alcoholdehydrogenase ") of short lactobacillus or Lactobacillus minor and corresponding aminoacid sequence thus can be available from: for lactobacillus kefir, Genbank accession number AAP94029GI:33112056 or SEQ ID NO:3; For short lactobacillus, Genbank accession number CAD66648GL28400789 or SEQ ID NO:1; And for Lactobacillus minor, SEQ ID NO:141.
In some embodiments, Ketoreductase polypeptides herein has many modifications of reference sequences (such as naturally occurring polypeptide or engineered polypeptide) to produce the ketoreductase character of improvement.In such an implementation, one or more amino acid of reference polypeptide sequence can be comprised to the quantity of the modification of aminoacid sequence, 2 or more amino acid, 3 or more amino acid, 4 or more amino acid, 5 or more amino acid, 6 or more amino acid, 8 or more amino acid, 10 or more, 15 or more amino acid or 20 an or more amino acid, reach 10% of amino acid whose total quantity, reach 10% of amino acid whose total quantity, reach 15% of amino acid whose total quantity, reach 20% of amino acid whose total quantity or reach 30% of amino acid whose total quantity.In some embodiments, the quantity producing the modification to naturally occurring polypeptide or engineered polypeptide of the ketoreductase character improved can comprise about 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-26,1-30,1-35 of reference sequences or about 1-40 residue modification.In some embodiments, the quantity of modification can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 amino-acid residue.Modification can comprise insertion, disappearance, replace or its combination.
In some embodiments, the aminoacid replacement comprised reference sequences is modified.The replacement that can produce the ketoreductase character of improvement can be positioned at one or more amino acid of reference enzyme sequence, 2 or more amino acid, 3 or more amino acid, 4 or more amino acid, 5 or more amino acid, 6 or more amino acid, 8 or more amino acid, 10 or more, 15 or more amino acid, or 20% of 10% of 10% of 20 or more amino acid, nearly amino acid whose total quantity, nearly amino acid whose total quantity, nearly amino acid whose total quantity, or nearly amino acid whose total quantity 30%.In some embodiments, the quantity producing the replacement to naturally occurring polypeptide or engineered polypeptide of the ketoreductase character improved can comprise about 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-26,1-30,1-35 of reference sequences or an about 1-40 aminoacid replacement.In some embodiments, the quantity of replacement can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 amino-acid residue.
In some embodiments, 3-ketone group tetramethylene sulfide substrate to reduce with it or to change into the stereoselectivity of (R)-3-hydroxy tetrahydro thiophene relevant by the character (contrast wild-type or another engineered polypeptide) of the improvement of Ketoreductase polypeptides.In some embodiments, substrate Stereoselective excessively can be reduced into product with the per-cent steric isomer at least about 65%, 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 99.99% by Ketoreductase polypeptides.
In some embodiments, Ketoreductase polypeptides can with compared with wild-type lactobacillus kefir or short lactobacillus or Lactobacillus minor KRED (SEQ ID NO:4 or 2 or 142), the per-cent steric isomer excessive (s.e) improved, is reduced into product by substrate Stereoselective.In some embodiments, substrate Stereoselective excessively can be reduced into product with the per-cent steric isomer at least about 70% by Ketoreductase polypeptides.
In some embodiments, Ketoreductase polypeptides of the present disclosure has the stereoselectivity of height, because substrate can be reduced into product to be greater than about 90% steric isomer excessive (s.e.) by ketoreductase.Have this High level of stereoselectivity optionally exemplary Ketoreductase polypeptides include but not limited to comprise corresponding SEQ ID NO:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106, 108, 110, 112, 126, 128, the polypeptide of the aminoacid sequence of 130 and 134.
In some embodiments, be enhanced in the speed (namely substrate conversion is speed or the ability of product by they) that Ketoreductase polypeptides of the present disclosure and wild-type (SEQ ID NO:4) contrast the enzymatic activity at them.Can with the speed exceeding wild-type the Exemplary polypeptide that substrate conversion is product included but not limited to comprise corresponding SEQ ID NO:6,8,10,12,14,16,18,20,22,24,26,30,32,38,40,42,44,46,48,50,52,54,56,58,60,64,66,68,70,74,76,78,80,86,88,90,92,104,106,110,112,124,126, the polypeptide of the aminoacid sequence of 130 and 134.
In some embodiments, Ketoreductase polypeptides of the present disclosure has High level of stereoselectivity selectivity, and can be greater than about 98%s.e. just substrate be reduced into product.Have this stereoselective Exemplary polypeptide include but not limited to comprise corresponding SEQ ID NO:6,8,10,18,20,22,24,26,28,30,34,36,38,40,42,50,52,54,58,62,66,70,72,76,78, the polypeptide of the aminoacid sequence of 80 and 134.
In some embodiments, Ketoreductase polypeptides of the present disclosure and wild-type contrast and are enhanced in their thermostability, and it is by high temperature, and the raising contrasting the speed of enzymatic activity with wild-type determined.The exemplary Ketoreductase polypeptides with the stability of improvement include but not limited to comprise corresponding SEQ ID NO:6,8,12,14,22,24,26,32,34,36,38,42,44,46,50,52,56,58,60,64,66,68,70,74,76,78,80,82,86,88,90,92,104,106,110,112, the polypeptide of the aminoacid sequence of 124 and 134.
In some embodiments, Ketoreductase polypeptides of the present disclosure has activity and the stability of the improvement exceeding wild-type, and can be greater than about 95%s.e. just substrate be reduced into product.The Exemplary polypeptide with this ability include but not limited to comprise corresponding SEQ DI NO:6,8,12,14,22,24,26,30,32,38,42,44,46,50,52,56,58,60,64,66,68,70,74,76,78,80,82,86,88,90,92,104,106,110, the polypeptide of the aminoacid sequence of 112 and 134.
In some embodiments, ketoreductase of the present disclosure can at room temperature with the Ketoreductase polypeptides of about 0.8-1.0g/L being less than at least 100g/L ketone group tetramethylene sulfide reduction or the product changing into correspondence 24 hours (such as about 20-24 hour) in.The Exemplary polypeptide with this ability include but not limited to comprise corresponding SEQ ID NO:6,8,10,12,14,18,20,22,24,26,30,32,38,40,42,44,46,48,50,52,54,56,58,60,64,66,68,70,74,76,78,80,82,86,88,90,92,104,106,110,112,126, the polypeptide of the aminoacid sequence of 130 and 134.
In some embodiments, ketoreductase of the present disclosure can at room temperature being less than 20 hours in (such as about 12-20 hour) with the Ketoreductase polypeptides of about 0.8-1.0g/L will at least reducing or change into product by 100g/L ketone group tetramethylene sulfide.The Exemplary polypeptide with this ability includes but not limited to SEQ ID NO:26,44,68 and 104.
Exemplary ketone reductase enzyme is provided with following table 2.Unless otherwise noted, following all sequences is derived from wild-type lactobacillus kefir ketoreductase sequence (SEQ ID NO:3 and 4).Each peptide sequence in table 2 is by SEQ ID NO nucleotide sequence coded had than the numbering few 1 in table, and namely the polypeptide of SEQID NO:4 is nucleotide sequence coded by SEQ ID NO:3.
Table 2: the list of sequence and character
In above table 2, in stereoselectivity row, " 0 " represents the about 61.0-71.9%s.e. of product (R)-3-hydroxy tetrahydro thiophene, "+" represents the about 80.0-89.9%s.e. of product, " ++ " represents the about 90.0-94.9%s.e. of product, " +++ " represents the about 95.0-97.9%s.e. of product, and " ++++" represents the about 98.0-98.0%s.e. of product, and " +++ ++ " represent product be greater than 99.0%s.e..In activity row, " 0 " represents and at room temperature transforms 100g/L ketone group tetramethylene sulfide completely and need 1.0-4.0g/L KRED being greater than in 24 hours; "+" represents that in 20-24 hour, at room temperature transform 100g/L ketone group tetramethylene sulfide completely needs 0.8-1.0g/L KRED; And " ++ " represents that at room temperature transforming 100g/L ketone group tetramethylene sulfide in 12-20 hour completely needs 0.8-1.0g/L KRED.In stability row, "-" shows the polypeptide of the thermostability than wild-type difference; " 0 " shows the polypeptide of the thermostability similar to wild-type; "+" shows the polypeptide of the thermostability of the improvement compared with wild-type; And " ++ " shows the polypeptide of the thermostability improved more compared with wild-type.
In some embodiments, the Ketoreductase polypeptides of improvement herein comprise with based on SEQ IDNO:2,4 or 142 reference sequences contrast, there is the aminoacid sequence of identity at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, in reference sequences, the amino-acid residue of corresponding 145 is polar residues, particularly Serine, condition is that Ketoreductase polypeptides has polar residues, particularly Serine at the residue place of corresponding 145.In some embodiments, Ketoreductase polypeptides and reference sequences contrast, and can have one or more residue difference at other amino-acid residue place.The difference contrasted with reference sequences can comprise replace, deletion and insertion.Difference can be non-conservative substitutions, the combination of guarding replacement or non-conservative and conservative replacement.In some embodiments, these Ketoreductase polypeptides optionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-26,1-30,1-35 or an about 1-40 difference at other amino-acid residue place.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 difference with reference sequences in the quantity of the difference at other amino-acid residue places.In some embodiments, the difference contrasted with reference sequences comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides of improvement comprises aminoacid sequence or its structural domain, such as the residue 90-211 of list type listed in corresponding SEQ ID NO:143 or SEQ ID NO:144 or SEQ ID NO:145.SEQ ID NO:145 is based on the wild-type amino acid sequence (SEQ ID NO:4) of lactobacillus kefir ketoreductase; SEQ ID NO:144 is based on the wild-type amino acid sequence (SEQ ID NO:2) of short lactobacillus ketoreductase; And SEQ ID NO:143 is based on the wild-type amino acid sequence (SEQ ID NO:142) of Lactobacillus minor ketoreductase.Based on SEQ ID NO:143,144 and 145 the ketoreductase of the list type residue that indicates corresponding X145 be polare Aminosaeren.
In some embodiments, Ketoreductase polypeptides comprises based on the list type of SEQ ID NO:143.144. or 145 or its structural domain (such as residue 90-211), and there is at the residue place of corresponding X145 the aminoacid sequence of polar residues particularly Serine, described Ketoreductase polypeptides can comprise further and is selected from following one or more features: the residue of corresponding X3 is polarity, acidity or aromatic moieties; The residue of corresponding X7 is nonpolar, polarity or restriction residue; The residue of corresponding X11 is aliphatics, nonpolar or polar residues; The residue of corresponding X16 is aliphatics or non-polar residue; The residue of corresponding X19 is nonpolar or aliphatic residue; The residue of corresponding X23 is nonpolar or aromatic moieties; The residue of corresponding X41 is aliphatics, nonpolar or polar residues; The residue of corresponding X45 is aliphatics, nonpolar or polar residues; The residue of corresponding X49 is alkaline residue; The residue of corresponding X57 is aliphatics or non-polar residue; The residue of corresponding X60 is aromatics, aliphatics, nonpolar or polar residues; The residue of corresponding X64 is aliphatics or non-polar residue; The residue of corresponding X72 is alkaline residue; The residue of corresponding X82 is nonpolar or polar residues; The residue of corresponding X94 is polarity, alkalescence, aliphatics or non-polar residue; The residue of corresponding X95 is nonpolar or aliphatic residue; The residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues; The residue of corresponding X97 is acid, alkaline or aliphatic residue; The residue of corresponding X106 is acidic residues; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties; The residue of corresponding X111 is aliphatics or non-polar residue; The residue of corresponding X117 is nonpolar or polar residues; The residue of corresponding X126 is aliphatics or non-polar residue; The residue of corresponding X127 is polarity or alkaline residue; The residue of corresponding X147 is aromatics, aliphatic residue, nonpolar or polar residues; The residue of corresponding X152 is polarity, aliphatics or non-polar residue; The residue of corresponding X157 is polarity or acidic residues; The residue of corresponding X163 is aliphatics or non-polar residue; The residue of corresponding X173 is acid or non-polar residue; The residue of corresponding X177 is alkaline residue; The residue of corresponding X192 is alkalescence or acidic residues; The residue of corresponding X194 is restriction, polarity, acidity, nonpolar or aliphatic residue; The residue of corresponding X198 is acid or non-polar residue; The residue of corresponding X200 is restriction, acid or alkaline residue; The residue of corresponding X206 is polarity or non-polar residue; The residue of corresponding X208 is polarity, restriction or alkaline residue; The residue of corresponding X210 is aliphatics, nonpolar or polar residues; The residue of corresponding X211 is alkalescence or acidic residues; The residue of corresponding X214 is nonpolar, aliphatics or polar residues; The residue of corresponding X217 is aromatics, aliphatics or non-polar residue; The residue of corresponding X223 is aliphatics or non-polar residue; And the residue of corresponding X226 is nonpolar or aliphatic residue.In some embodiments, the residue of corresponding X94 is not aromatic moieties, Histidine or glycine.In some embodiments, the residue of corresponding X96 is not aromatic moieties, glycine, Isoleucine or halfcystine.In some embodiments, comprise corresponding SEQID NO:143, one or more in residue that X that the polypeptide of aminoacid sequence of list type (or its structural domain) listed by 144 or 145 can have conservative variants does not point out.In some embodiments, conservative variants can be in 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-26,1-30,1-35 of other amino-acid residues do not defined by above X or about 1-40 sudden change.In some embodiments, the quantity of sudden change can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 amino-acid residue.
In some embodiments, comprise based on the polypeptide of the aminoacid sequence of the list type provided in SEQ ID NO:143,144 or 145 and SEQ ID NO:4,2 or 142 reference sequences contrast, one or more conservative variants can be had.Exemplary conservative replaces and comprises amino-acid substitution, such as, but not limited to: use another aliphatic residue, such as α-amino-isovaleric acid replaces the residue (I) of corresponding X19; With using another non-polar residue, the residue (I) of corresponding X23 replaced by such as phenylalanine; Use another alkaline residue, such as arginine replaces the residue (K) of corresponding X49; Use another aliphatic residue, such as α-amino-isovaleric acid replaces the residue (I) of corresponding X57; Use another basic aminoacids, such as arginine replaces the residue (K) of corresponding X72; Use another aliphatic amino acid, such as α-amino-isovaleric acid replaces the residue (A) of corresponding X94; Use another aliphatic residue, such as α-amino-isovaleric acid replaces the residue (A) of corresponding X95; Use another basic aminoacids, such as arginine replaces the residue (K) of corresponding X97; Use another acidic amino acid, the residue (E) of the corresponding X106 of such as aspartic acid; Nonpolar or aliphatic amino acid with another, the residue (L) of corresponding X111 replaced by such as methionine(Met); Use another nonpolar amino acid, such as leucine displacement residue X147 (F); Use another basic aminoacids, such as arginine replaces the residue (K) of corresponding X177; Use another basic aminoacids, such as arginine replaces the residue (K) of corresponding X192; Use another nonpolar amino acid, such as α-amino-isovaleric acid replaces the residue (M) of corresponding X214; Use another nonpolar amino acid, the residue (I) of corresponding X217 replaced by such as phenylalanine; Nonpolar or aliphatic amino acid with another, such as α-amino-isovaleric acid replaces the residue (I) of corresponding X223; Such as, and nonpolar or aliphatic amino acid with another, the residue (I) of corresponding X226 replaced by leucine.
In some embodiments, Ketoreductase polypeptides comprise based on SEQ ID NO:143,144 or 145 list type or its structural domain (such as residue 90-211), and there is at the residue place of corresponding X145 the aminoacid sequence of polar residues particularly Serine, described Ketoreductase polypeptides can comprise further and is selected from following one or more features: the residue of corresponding X3 is l-asparagine, aspartic acid or tyrosine; The residue of corresponding X7 is glycine, Histidine, Serine or l-asparagine; The residue of corresponding X11 is Isoleucine or Threonine; The residue of corresponding X16 is Threonine, L-Ala, α-amino-isovaleric acid or glycine; The residue of corresponding X19 is Isoleucine or α-amino-isovaleric acid; The residue of corresponding X23 is Isoleucine or phenylalanine; The residue of corresponding X41 is Serine, L-Ala or α-amino-isovaleric acid; The residue of corresponding X45 is L-glutamic acid or glycine; The residue of corresponding X49 is Methionin or arginine, particularly arginine; The residue of corresponding X57 is Isoleucine or α-amino-isovaleric acid; The residue of corresponding X60 is phenylalanine, α-amino-isovaleric acid or Threonine; The residue of corresponding X64 is L-Ala, Serine or Threonine; The residue of corresponding X72 is Methionin or arginine, particularly arginine; The residue of corresponding X82 is glycine or Serine; The residue of corresponding X94 is L-Ala, α-amino-isovaleric acid, Threonine, Serine or arginine; The residue of corresponding X95 is α-amino-isovaleric acid or L-Ala; The residue of corresponding X96 is l-asparagine, Serine, proline(Pro), L-Ala or L-glutamic acid; The residue of corresponding X97 is Methionin, arginine or leucine; The residue of corresponding X106 is L-glutamic acid or aspartic acid; The residue of corresponding X108 is arginine or Histidine; The residue of corresponding X111 is leucine or methionine(Met); The residue of corresponding X117 is glycine or Serine; The residue of corresponding X126 is Isoleucine or α-amino-isovaleric acid; The residue of corresponding X127 is glutamine or arginine; The residue of corresponding X147 is phenylalanine, leucine or Serine; The residue of corresponding X152 is Threonine, Serine or methionine(Met); The residue of corresponding X157 is l-asparagine, glutamine, Threonine, Serine or aspartic acid; The residue of corresponding X163 is α-amino-isovaleric acid or Isoleucine; The residue of corresponding X173 is aspartic acid or glycine; The residue of corresponding X177 is Methionin or arginine; The residue of corresponding X192 is Methionin, arginine or L-glutamic acid; The residue of corresponding X194 is proline(Pro), glycine, aspartic acid, arginine or leucine; The residue of corresponding X198 is aspartic acid or glycine; The residue of corresponding X200 is proline(Pro), L-glutamic acid or Methionin; The residue of corresponding X206 is methionine(Met) or glutamine; The residue of corresponding X208 is glutamine, Histidine or arginine; The residue of corresponding X210 is Threonine or L-Ala; The residue of corresponding X211 is Methionin or L-glutamic acid; The residue of corresponding X214 is methionine(Met), α-amino-isovaleric acid or Threonine or Serine, particularly α-amino-isovaleric acid or Threonine; The residue of corresponding X217 is Isoleucine or phenylalanine; The residue of corresponding X223 is Isoleucine or α-amino-isovaleric acid; And the residue of corresponding X226 is Isoleucine or α-amino-isovaleric acid.In some embodiments, comprise corresponding SEQ ID NO:143, one or more in residue that X that the polypeptide of aminoacid sequence of list type (or its structural domain) listed by 144 or 145 can have conservative variants does not point out.In some embodiments, conservative variants can be in 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-26,1-30,1-35 of other amino-acid residues do not defined by above X or about 1-40 sudden change.In some embodiments, the quantity of sudden change can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 amino-acid residue.
In some embodiments, Ketoreductase polypeptides comprises based on the list type of SEQ ID NO:143.144. or 145 or its structural domain (such as residue 90-211), and there is at the residue place of corresponding X145 the aminoacid sequence of polar residues particularly Serine, it is one or more or at least whole that described Ketoreductase polypeptides can comprise in following characteristics further: the residue of corresponding X7 is nonpolar, polarity or restriction residue; The residue of corresponding X94 is polarity, alkalescence, aliphatics or non-polar residue; The residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties; The residue of corresponding X117 is nonpolar or polar residues; The residue of corresponding X157 is polarity or acidic residues; The residue of corresponding X173 is acid or non-polar residue; The residue of corresponding X206 is polarity or non-polar residue; And the residue of corresponding X223 is aliphatics or non-polar residue.In some embodiments, the amino-acid residue of corresponding X145 is Serine.In some embodiments, except above feature, Ketoreductase polypeptides can comprise that to be selected from following feature one or more further: the residue of corresponding X3 is polarity, acidity or aromatic moieties; The residue of corresponding X11 is aliphatics, nonpolar or polar residues; The residue of corresponding X16 is aliphatics or non-polar residue; The residue of corresponding X19 is nonpolar or aliphatic residue; The residue of corresponding X23 is nonpolar or aromatic moieties; The residue of corresponding X41 is aliphatics, nonpolar or polar residues; The residue of corresponding X45 is glycine, aliphatics, nonpolar or polar residues; The residue of corresponding X49 is alkaline residue; The residue of corresponding X57 is aliphatics or non-polar residue; The residue of corresponding X60 is aromatics, aliphatics, nonpolar or polar residues; The residue of corresponding X64 is aliphatics or non-polar residue; The residue of corresponding X72 is alkaline residue; The residue of corresponding X82 is nonpolar or polar residues; The residue of corresponding X95 is nonpolar or aliphatic residue; The residue of corresponding X97 is acid, alkaline or aliphatic residue; The residue of corresponding X106 is acidic residues; The residue of corresponding X111 is aliphatics or non-polar residue; The residue of corresponding X126 is aliphatics or non-polar residue; The residue of corresponding X127 is polarity or alkaline residue; The residue of corresponding X147 is aromatics, aliphatic residue, nonpolar or polar residues; The residue of corresponding X152 is polarity, aliphatics or non-polar residue; The residue of corresponding X163 is aliphatics or non-polar residue; The residue of corresponding X177 is alkaline residue; The residue of corresponding X192 is alkalescence or acidic residues; The residue of corresponding X194 is restriction, polarity, nonpolar, acid or aliphatic residue; The residue of corresponding X198 is acid or non-polar residue; The residue of corresponding X200 is restriction, acid or alkaline residue; The residue of corresponding X208 is polarity, restriction or alkaline residue; The residue of corresponding X210 is aliphatics, nonpolar or polar residues; The residue of corresponding X211 is alkalescence or acidic residues; The residue of corresponding X214 is nonpolar, aliphatics or polar residues; The residue of corresponding X217 is aromatics, aliphatics or non-polar residue; And the residue of corresponding X226 is nonpolar or aliphatic residue.In some embodiments, comprise corresponding SEQ ID NO:143, one or more in residue that X that the polypeptide of aminoacid sequence of list type (or its structural domain) listed by 144 or 145 can have conservative variants does not point out.In some embodiments, conservative variants can be in 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-26,1-30,1-35 of other amino-acid residues do not defined by above X or about 1-40 sudden change.In some embodiments, the quantity of sudden change can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 amino-acid residue.
In some embodiments, Ketoreductase polypeptides comprises based on the list type of SEQ ID NO:143.144. or 145 or its structural domain (such as residue 90-211), and there is at the residue place of corresponding X145 the aminoacid sequence of polar residues particularly Serine, described Ketoreductase polypeptides can comprise that to be selected from following feature one or more or at least whole further: the residue of corresponding X7 is glycine, Histidine, Serine or l-asparagine; The residue of corresponding X94 is L-Ala, α-amino-isovaleric acid, Threonine, Serine or arginine; The residue of corresponding X96 is l-asparagine, Serine, proline(Pro), L-Ala or L-glutamic acid; The residue of corresponding X108 is arginine or Histidine; The residue of corresponding X117 is glycine or Serine; The residue of corresponding X157 is l-asparagine, glutamine, Threonine, Serine or aspartic acid; The residue of corresponding X173 is aspartic acid or glycine; The residue of corresponding X206 is methionine(Met) or glutamine; And the residue of corresponding X223 is Isoleucine or α-amino-isovaleric acid.In some embodiments, except above feature, Ketoreductase polypeptides can comprise that to be selected from following feature one or more further: the residue of corresponding X3 is l-asparagine, aspartic acid or tyrosine; The residue of corresponding X11 is Isoleucine or Threonine; The residue of corresponding X16 is Threonine, L-Ala, α-amino-isovaleric acid or glycine; The residue of corresponding X19 is Isoleucine or α-amino-isovaleric acid; The residue of corresponding X23 is Isoleucine or phenylalanine; The residue of corresponding X41 is Serine, L-Ala or α-amino-isovaleric acid; The residue of corresponding X45 is L-glutamic acid or glycine; The residue of corresponding X49 is Methionin or arginine, particularly arginine; The residue of corresponding X57 is Isoleucine or α-amino-isovaleric acid; The residue of corresponding X60 is phenylalanine, α-amino-isovaleric acid or Threonine; The residue of corresponding X64 is L-Ala, Serine or Threonine; The residue of corresponding X72 is Methionin or arginine, particularly arginine; The residue of corresponding X82 is glycine or Serine; The residue of corresponding X95 is α-amino-isovaleric acid or L-Ala; The residue of corresponding X97 is Methionin, arginine or leucine; The residue of corresponding X106 is L-glutamic acid or aspartic acid; The residue of corresponding X111 is leucine or methionine(Met); The residue of corresponding X126 is Isoleucine or α-amino-isovaleric acid; The residue of corresponding X127 is glutamine or arginine; The residue of corresponding X147 is phenylalanine, leucine or Serine; The residue of corresponding X152 is Threonine, Serine or methionine(Met); The residue of corresponding X163 is α-amino-isovaleric acid or Isoleucine; The residue of corresponding X177 is Methionin or arginine; The residue of corresponding X192 is Methionin, arginine or L-glutamic acid; The residue of corresponding X194 is proline(Pro), glycine, aspartic acid, arginine or leucine; The residue of corresponding X198 is aspartic acid or glycine; The residue of corresponding X200 is proline(Pro), L-glutamic acid or Methionin; The residue of corresponding X208 is glutamine, Histidine or arginine; The residue of corresponding X210 is Threonine or L-Ala; The residue of corresponding X211 is Methionin or L-glutamic acid; The residue of corresponding X214 is methionine(Met), α-amino-isovaleric acid or Threonine or Serine, particularly α-amino-isovaleric acid or Threonine; The residue of corresponding X217 is Isoleucine or phenylalanine; And the residue of corresponding X226 is Isoleucine or α-amino-isovaleric acid.In some embodiments, comprise corresponding SEQ ID NO:143, one or more in residue that X that the polypeptide of aminoacid sequence of list type (or its structural domain) listed by 144 or 145 can have conservative variants does not point out.In some embodiments, conservative variants can be in 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-26,1-30,1-35 of other amino-acid residues do not defined by above X or about 1-40 sudden change.In some embodiments, the quantity of sudden change can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 other amino-acid residue.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X3 is polarity, acidity or aromatic moieties, particularly tyrosine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X7 is nonpolar, polarity or restriction residue, particularly Serine, Histidine or arginine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X11 is aliphatics, nonpolar or polar residues.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X16 is aliphatics or non-polar residue, particularly L-Ala or glycine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X19 is nonpolar or aliphatic residue, particularly L-Ala or glycine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X23 is nonpolar or aromatic moieties, particularly phenylalanine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X41 is aliphatics, nonpolar or polar residues, particularly Serine, L-Ala or α-amino-isovaleric acid.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X45 is glycine, aliphatics, nonpolar or polar residues, particularly L-glutamic acid or glycine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X49 is alkaline residue, particularly arginine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X57 is aliphatics or non-polar residue, particularly Isoleucine or α-amino-isovaleric acid.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X60 is aromatics, aliphatics, nonpolar or polar residues, particularly phenylalanine, α-amino-isovaleric acid or Threonine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X64 is aliphatics or non-polar residue, particularly L-Ala, Serine or Threonine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X72 is alkaline residue, particularly L-Ala, Serine or Threonine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X82 is nonpolar or polar residues, particularly glycine or Serine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X94 is polarity, alkalescence, aliphatics or non-polar residue, particularly α-amino-isovaleric acid, Threonine or arginine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ IDNO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X95 is non-polar residue or aliphatic residue, particularly L-Ala.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues, particularly l-asparagine, Serine, proline(Pro), L-Ala or L-glutamic acid.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X97 is acid, alkaline or aliphatic residue, particularly Methionin, arginine or leucine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X106 is acidic residues, particularly aspartic acid.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly arginine or Histidine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X111 is aliphatics or non-polar residue, particularly methionine(Met).In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X117 is nonpolar or polar residues, particularly Serine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X126 is aliphatics or non-polar residue, particularly α-amino-isovaleric acid.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X127 is polarity or alkaline residue, particularly glutamine or arginine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X147 is aromatics, aliphatic residue, nonpolar or polar residues, particularly phenylalanine, leucine or Serine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X152 is polarity, aliphatics or non-polar residue, nonpolar or polar residues, particularly Threonine, Serine or methionine(Met).In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X157 is polarity or alkaline residue, particularly l-asparagine, Threonine, Serine or aspartic acid.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X163 is aliphatics or non-polar residue, particularly α-amino-isovaleric acid or Isoleucine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X173 is acid or non-polar residue, particularly aspartic acid or glycine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X177 is alkaline residue, particularly arginine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X192 is alkalescence or acidic residues, particularly Methionin, arginine or L-glutamic acid.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X194 is restriction, polarity, nonpolar, acid or aliphatic residue, particularly proline(Pro), glycine, aspartic acid, arginine or leucine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X198 is acid or non-polar residue, particularly aspartic acid or glycine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X200 is restriction, acid or alkaline residue, particularly proline(Pro), L-glutamic acid or Methionin.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X200 is restriction, acid or alkaline residue, particularly proline(Pro), L-glutamic acid or Methionin.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X206 is polarity or non-polar residue, particularly methionine(Met) or glutamine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X208 is polarity, restriction or alkaline residue, particularly glutamine, Histidine or arginine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X210 is aliphatics, nonpolar or polar residues, particularly Threonine or L-Ala.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X211 is alkalescence or acidic residues, particularly Methionin or L-glutamic acid.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X214 is nonpolar, aliphatics or polar residues, particularly α-amino-isovaleric acid or Threonine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X217 is aromatics, aliphatics or non-polar residue, particularly Isoleucine or phenylalanine.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X223 is aliphatics or non-polar residue, particularly Isoleucine or α-amino-isovaleric acid.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X226 is nonpolar or aliphatic residue, particularly Isoleucine or α-amino-isovaleric acid.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X145 is polar residues, particularly Serine, and the residue of corresponding X228 is nonpolar or aliphatic residue, particularly α-amino-isovaleric acid or L-Ala.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Threonine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X7 is nonpolar, polarity or restriction residue, particularly Serine, Histidine or l-asparagine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Threonine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), and wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X94 is polarity, alkalescence, aliphatics or non-polar residue; Particularly α-amino-isovaleric acid, Threonine or arginine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Threonine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues, particularly l-asparagine, proline(Pro), L-Ala or L-glutamic acid; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Threonine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Threonine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X173 is acid or non-polar residue, particularly glycine; And the residue of corresponding X157 is polarity or acidic residues, particularly Threonine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polarity or acidic residues, particularly Threonine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X7 is nonpolar, polarity or restriction residue, particularly Serine, Histidine or l-asparagine; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Threonine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X7 is nonpolar, polarity or restriction residue, particularly Serine, Histidine or l-asparagine; The residue of corresponding X94 is polarity, alkalescence, aliphatics or non-polar residue; Particularly α-amino-isovaleric acid, Threonine or arginine; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Threonine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X7 is nonpolar, polarity or restriction residue, particularly Serine, Histidine or l-asparagine; The residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues, particularly l-asparagine, proline(Pro), L-Ala or L-glutamic acid; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Threonine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X7 is nonpolar, polarity or restriction residue, particularly Serine, Histidine or l-asparagine; The residue of corresponding X94 is polarity, alkalescence, aliphatics or non-polar residue; Particularly α-amino-isovaleric acid, Threonine or arginine; The residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues, particularly l-asparagine, proline(Pro), L-Ala or L-glutamic acid; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Threonine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polarity or acidic residues, particularly Threonine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X7 is nonpolar, polarity or restriction residue, particularly Serine, Histidine or l-asparagine; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polarity or acidic residues, particularly Threonine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X7 is nonpolar, polarity or restriction residue, particularly Serine, Histidine or l-asparagine; The residue of corresponding X94 is polarity, alkalescence, aliphatics or non-polar residue; Particularly α-amino-isovaleric acid, Threonine or arginine; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polarity or acidic residues, particularly Threonine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X7 is nonpolar, polarity or restriction residue, particularly Serine, Histidine or l-asparagine; The residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues, particularly l-asparagine, proline(Pro), L-Ala or L-glutamic acid; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polarity or acidic residues, particularly Threonine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X7 is nonpolar, polarity or restriction residue, particularly Serine, Histidine or l-asparagine; The residue of corresponding X94 is polarity, alkalescence, aliphatics or non-polar residue; Particularly α-amino-isovaleric acid, Threonine or arginine; The residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues, particularly l-asparagine, proline(Pro), L-Ala or L-glutamic acid; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polarity or acidic residues, particularly Threonine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polarity or acidic residues, particularly Threonine; The residue of corresponding X173 is acid or non-polar residue, particularly glycine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X7 is nonpolar, polarity or restriction residue, particularly Serine, Histidine or l-asparagine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polarity or acidic residues, particularly Threonine; The residue of corresponding X173 is acid or non-polar residue, particularly glycine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X7 is nonpolar, polarity or restriction residue, particularly Serine, Histidine or l-asparagine; The residue of corresponding X94 is polarity, alkalescence, aliphatics or non-polar residue; Particularly α-amino-isovaleric acid, Threonine or arginine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polarity or acidic residues, particularly Threonine; The residue of corresponding X173 is acid or non-polar residue, particularly glycine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X7 is nonpolar, polarity or restriction residue, particularly Serine, Histidine or l-asparagine; The residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues, particularly l-asparagine, proline(Pro), L-Ala or L-glutamic acid; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polarity or acidic residues, particularly Threonine; The residue of corresponding X173 is acid or non-polar residue, particularly glycine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQ ID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the Ketoreductase polypeptides improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), wherein said aminoacid sequence has at least following characteristics: the residue of corresponding X7 is nonpolar, polarity or restriction residue, particularly Serine, Histidine or l-asparagine; The residue of corresponding X94 is polarity, alkalescence, aliphatics or non-polar residue; Particularly α-amino-isovaleric acid, Threonine or arginine; The residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues, particularly l-asparagine, proline(Pro), L-Ala or L-glutamic acid; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polarity or acidic residues, particularly Threonine; The residue of corresponding X173 is acid or non-polar residue, particularly glycine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, ketoreductase can have aliphatic residue at the residue place of corresponding X223, particularly α-amino-isovaleric acid extraly.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein aminoacid sequence with have above feature based on SEQID NO:2,4 or 142 reference sequences contrast, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprises and the SEQ IDNO:6 be selected from as listed in table 2, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106, 108, 110, 112, 126, 128, the reference sequences of 130 and 134 has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, the aminoacid sequence of the identity of 98% or 99%, the Ketoreductase polypeptides aminoacid sequence of wherein said improvement comprises any one group of aminoacid replacement combination of specifying provided in table 2.In some embodiments, contrast with reference sequences, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 difference at other amino-acid residue places.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase of improvement comprise have corresponding SEQ ID NO:6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42,44,46,48,50,52,54,56,58,60,62,64,66,68,70,72,74,76,78,80,82,86,88,90,92,94,96,100,102,104,106,108,110,112,126,128, the polypeptide of the aminoacid sequence of 130 and 134.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X72 is alkaline residue, particularly arginine; The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:104, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:104 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly Serine; The residue of corresponding X108 is restriction residue, particularly Histidine; The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:44, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:44 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly Serine; The residue of corresponding X49 is alkaline residue, particularly arginine; The residue of corresponding X111 is non-polar residue, particularly methionine(Met); The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; The residue of corresponding X173 is non-polar residue, particularly glycine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:68, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:68 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly Serine; The residue of corresponding X94 is nonpolar or polar residues, particularly Threonine; The residue of corresponding X97 is nonpolar or aliphatic residue, particularly leucine; The residue of corresponding X108 is restriction residue, particularly Histidine; The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; The residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid; And the residue of corresponding X226 is aliphatic residue, particularly leucine.In some embodiments, contrast with the reference sequences of SEQ ID NO:42, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQID NO:42 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly Serine; The residue of corresponding X94 is nonpolar or polar residues, particularly Threonine; The residue of corresponding X96 is restriction or acidic residues, particularly proline(Pro); The residue of corresponding X108 is restriction residue, particularly Histidine; The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:24, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:24 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly Serine; The residue of corresponding X94 is nonpolar or polar residues, particularly Threonine; The residue of corresponding X108 is restriction residue, particularly Histidine; The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; The residue of corresponding X206 is polar residues, particularly glutamine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:38, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:38 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly Serine; The residue of corresponding X94 is nonpolar or polar residues, particularly Threonine; The residue of corresponding X96 is restriction or acidic residues, particularly L-glutamic acid; The residue of corresponding X108 is restriction residue, particularly Histidine; The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:32, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:32 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly arginine; The residue of corresponding X94 is nonpolar or polar residues, particularly Threonine; The residue of corresponding X106 is acidic residues, particularly aspartic acid; The residue of corresponding X108 is restriction residue, particularly Histidine; The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:8, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:8 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X3 is aromatic moieties, particularly tyrosine; The residue of corresponding X7 is polar residues, particularly arginine; The residue of corresponding X94 is nonpolar or polar residues, particularly Threonine; The residue of corresponding X108 is restriction residue, particularly Histidine; The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:134, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:134 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly arginine; The residue of corresponding X94 is nonpolar or polar residues, particularly Threonine; The residue of corresponding X108 is restriction residue, particularly Histidine; The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; The residue of corresponding X208 is restriction residue, particularly Histidine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:6, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:6 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly arginine; The residue of corresponding X94 is nonpolar or polar residues, particularly Threonine; The residue of corresponding X96 is restriction residue, particularly proline(Pro); The residue of corresponding X108 is restriction residue, particularly Histidine; The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; The residue of corresponding X206 is polar residues, particularly glutamine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:26, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:26 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly arginine; The residue of corresponding X108 is restriction residue, particularly Histidine; The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; The residue of corresponding X206 is polar residues, particularly glutamine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:52, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:52 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly arginine; The residue of corresponding X96 is restriction residue, particularly proline(Pro); The residue of corresponding X108 is restriction residue, particularly Histidine; The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; The residue of corresponding X206 is polar residues, particularly glutamine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:50, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQID NO:50 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly arginine; The residue of corresponding X16 is nonpolar or aliphatic residue, particularly glycine; The residue of corresponding X96 is restriction residue, particularly proline(Pro); The residue of corresponding X108 is restriction residue, particularly Histidine; The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; The residue of corresponding X206 is polar residues, particularly glutamine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:76, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:76 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly arginine; The residue of corresponding X16 is nonpolar or aliphatic residue, particularly α-amino-isovaleric acid; The residue of corresponding X94 is nonpolar or polar residues, particularly Threonine; The residue of corresponding X96 is restriction residue, particularly proline(Pro); The residue of corresponding X108 is restriction residue, particularly Histidine; The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; The residue of corresponding X206 is polar residues, particularly glutamine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:80, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:80 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly arginine; The residue of corresponding X16 is nonpolar or aliphatic residue, particularly glycine; The residue of corresponding X94 is nonpolar or polar residues, particularly Threonine; The residue of corresponding X96 is restriction residue, particularly proline(Pro); The residue of corresponding X108 is restriction residue, particularly Histidine; The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; The residue of corresponding X206 is polar residues, particularly glutamine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:78, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:78 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly arginine; The residue of corresponding X94 is nonpolar or polar residues, particularly Threonine; The residue of corresponding X96 is restriction residue, particularly proline(Pro); The residue of corresponding X108 is restriction residue, particularly Histidine; The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; The residue of corresponding X194 is non-polar residue, particularly glycine; The residue of corresponding X206 is polar residues, particularly glutamine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:30, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:30 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly arginine; The residue of corresponding X94 is nonpolar or polar residues, particularly Threonine; The residue of corresponding X96 is restriction residue, particularly proline(Pro); The residue of corresponding X108 is restriction residue, particularly Histidine; The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; The residue of corresponding X194 is alkaline residue, particularly arginine; The residue of corresponding X206 is polar residues, particularly glutamine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:22, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:22 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly arginine; The residue of corresponding X49 is alkaline residue, particularly arginine; The residue of corresponding X111 is nonpolar or aliphatic residue, particularly methionine(Met); The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; The residue of corresponding X173 is non-polar residue, particularly glycine; The residue of corresponding X206 is polar residues, particularly glutamine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:70, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:70 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly arginine; The residue of corresponding X49 is alkaline residue, particularly arginine; The residue of corresponding X96 is restriction residue, particularly proline(Pro); The residue of corresponding X111 is nonpolar or aliphatic residue, particularly methionine(Met); The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; The residue of corresponding X173 is non-polar residue, particularly glycine; The residue of corresponding X206 is polar residues, particularly glutamine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:66, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:66 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprises based on SEQ ID NO:143, the list type of 144 or 145 or the aminoacid sequence of its structural domain (such as residue 90-211), it has at least following characteristics: the residue of corresponding X7 is polar residues, particularly arginine; The residue of corresponding X49 is alkaline residue, particularly arginine; The residue of corresponding X94 is polar residues, particularly Threonine; The residue of corresponding X96 is restriction residue, particularly proline(Pro); The residue of corresponding X111 is nonpolar or aliphatic residue, particularly methionine(Met); The residue of corresponding X117 is polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; The residue of corresponding X157 is polar residues, particularly Threonine; The residue of corresponding X173 is non-polar residue, particularly glycine; The residue of corresponding X206 is polar residues, particularly glutamine; And the residue of corresponding X223 is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, contrast with the reference sequences of SEQ ID NO:58, Ketoreductase polypeptides can additionally have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 residue difference at other residue positions.In some embodiments, the quantity of the difference at other amino-acid residue places can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with above feature, and wherein said aminoacid sequence and SEQ ID NO:58 have the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase of improvement comprise have corresponding SEQ ID NO:143,144 or 145 the region of residue 90-211 of list type or the aminoacid sequence of structural domain, the residue of the corresponding X145 wherein in structural domain is polar residues.In some embodiments, corresponding SEQ IDNO:143,144 or 145 the region of residue 90-211 of list type or structural domain at the residue place of corresponding X145, there is Serine.In some embodiments, with based on SEQ ID NO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein said aminoacid sequence with there is above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 90-211 of reference sequences, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, there is the structural domain of corresponding residue 90-211, and at the residue place of corresponding X145, there is polar residues, particularly the Ketoreductase polypeptides of Serine can comprise further and is selected from following one or more features: the residue of corresponding X94 is polarity, alkalescence, aliphatics or non-polar residue; The residue of corresponding X95 is nonpolar or aliphatic residue; The residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues; The residue of corresponding X97 is acid, alkaline or aliphatic residue; The residue of corresponding X106 is acidic residues; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties; The residue of corresponding X111 is aliphatics or non-polar residue; The residue of corresponding X117 is nonpolar or polar residues; The residue of corresponding X126 is aliphatics or non-polar residue; The residue of corresponding X127 is polarity or alkaline residue; The residue of corresponding X147 is aromatics, aliphatic residue, nonpolar or polar residues; The residue of corresponding X152 is polarity, aliphatics or non-polar residue; The residue of corresponding X157 is polarity or acidic residues; The residue of corresponding X163 is aliphatics or non-polar residue; The residue of corresponding X173 is acid or non-polar residue; The residue of corresponding X177 is alkaline residue; The residue of corresponding X192 is alkalescence or acidic residues; The residue of corresponding X194 is restriction, polarity, nonpolar, acid or aliphatic residue; The residue of corresponding X198 is acid or non-polar residue; The residue of corresponding X200 is restriction, acid or alkaline residue; The residue of corresponding X206 is polarity or non-polar residue; The residue of corresponding X208 is polarity, restriction or alkaline residue; The residue of corresponding X210 is aliphatics, nonpolar or polar residues; And the residue of corresponding X211 is alkalescence or acidic residues.In some embodiments, the residue of corresponding X94 is not aromatic moieties, Histidine or glycine.In some embodiments, the residue of corresponding X96 is not aromatic moieties, glycine, Isoleucine or halfcystine.In some embodiments, with based on SEQ ID NO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have about 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.
In some embodiments, with SEQ ID NO:4,2 or 142 Amino acid sequences alignment, have corresponding based on SEQ ID NO:143,144 or 145 the structural domain of residue 90-211 of list type or the Ketoreductase polypeptides in region can have one or more conservative variants.Exemplary conservative replaces and comprises amino-acid substitution, such as, but not limited to: use another aliphatic amino acid, such as α-amino-isovaleric acid replaces the residue (A) of corresponding X94; Nonpolar or aliphatic residue with another, the residue (V) of the corresponding X95 of such as alanine substitution; Use another basic aminoacids, such as arginine replaces the residue (K) of corresponding X97; Use another acidic amino acid, the residue (E) of the corresponding X106 of such as aspartic acid; Nonpolar or aliphatic amino acid with another, the residue (L) of corresponding X111 replaced by such as methionine(Met); Use another nonpolar amino acid, such as leucine displacement residue X147 (F); Use another basic aminoacids, such as arginine replaces the residue (K) of corresponding X177; Use another basic aminoacids, such as arginine replaces the residue (K) of corresponding X192.
In some embodiments, there is the structural domain of corresponding residue 90-211, and at the residue place of corresponding X145, there is polar residues, particularly the Ketoreductase polypeptides of Serine can comprise further and is selected from following one or more features: the residue of corresponding X94 is L-Ala, α-amino-isovaleric acid, Threonine, Serine or arginine; The residue of corresponding X95 is α-amino-isovaleric acid or L-Ala; The residue of corresponding X96 is l-asparagine, Serine, proline(Pro), L-Ala or L-glutamic acid; The residue of corresponding X97 is Methionin, arginine or leucine; The residue of corresponding X106 is L-glutamic acid or aspartic acid; The residue of corresponding X108 is arginine or Histidine; The residue of corresponding X111 is leucine or methionine(Met); The residue of corresponding X117 is glycine or Serine; The residue of corresponding X126 is Isoleucine or α-amino-isovaleric acid; The residue of corresponding X127 is glutamine or arginine; The residue of corresponding X147 is phenylalanine, leucine or Serine; The residue of corresponding X152 is Threonine, Serine or methionine(Met); The residue of corresponding X157 is l-asparagine, glutamine, Threonine, Serine or aspartic acid; The residue of corresponding X163 is α-amino-isovaleric acid or Isoleucine; The residue of corresponding X173 is aspartic acid or glycine; The residue of corresponding X177 is Methionin or arginine; The residue of corresponding X192 is Methionin, arginine or L-glutamic acid; The residue of corresponding X194 is proline(Pro), glycine, aspartic acid, arginine or leucine; The residue of corresponding X198 is aspartic acid or glycine; The residue of corresponding X200 is proline(Pro), L-glutamic acid or Methionin; The residue of corresponding X206 is methionine(Met) or glutamine; The residue of corresponding X208 is glutamine, Histidine or arginine; The residue of corresponding X210 is Threonine or L-Ala; And the residue of corresponding X211 is Methionin or L-glutamic acid.In some embodiments, with based on SEQ ID NO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.
In some embodiments, there is the structural domain of corresponding residue 90-211, and at the residue place of corresponding X145, there is polar residues, particularly the Ketoreductase polypeptides of Serine can comprise that to be selected from following feature one or more or at least whole further: the residue of corresponding X94 is polarity, alkalescence, aliphatics or non-polar residue; The residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties; The residue of corresponding X117 is nonpolar or polar residues; The residue of corresponding X157 is polarity or acidic residues; The residue of corresponding X173 is acid or non-polar residue; And the residue of corresponding X206 is polarity or non-polar residue.In some embodiments, except above feature, described structural domain or region can comprise that to be selected from following feature one or more further: the residue of corresponding X97 is acid, alkalescence or aliphatic residue; The residue of corresponding X106 is acidic residues; The residue of corresponding X111 is aliphatics or non-polar residue; The residue of corresponding X126 is aliphatics or non-polar residue; The residue of corresponding X127 is polarity or alkaline residue; The residue of corresponding X147 is aromatics, aliphatic residue, nonpolar or polar residues; The residue of corresponding X152 is polarity, aliphatics or non-polar residue; The residue of corresponding X163 is aliphatics or non-polar residue; The residue of corresponding X177 is alkaline residue; The residue of corresponding X192 is alkalescence or acidic residues; The residue of corresponding X194 is restriction, polarity, nonpolar, acid or aliphatic residue; The residue of corresponding X198 is acid or non-polar residue; The residue of corresponding X200 is restriction, acid or alkaline residue; The residue of corresponding X208 is polarity, restriction or alkaline residue; The residue of corresponding X210 is aliphatics, nonpolar or polar residues; And the residue of corresponding X211 is alkalescence or acidic residues.In some embodiments, with based on SEQ ID NO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.
In some embodiments, there is the structural domain of corresponding residue 90-211, and at the residue place of corresponding X145, there is polar residues, particularly the Ketoreductase polypeptides of Serine can comprise that to be selected from following feature one or more or at least whole further: the residue of corresponding X94 is L-Ala, α-amino-isovaleric acid, Threonine, Serine or arginine; The residue of corresponding X96 is l-asparagine, Serine, proline(Pro), L-Ala or L-glutamic acid; The residue of corresponding X108 is arginine or Histidine; The residue of corresponding X117 is glycine or Serine; The residue of corresponding X157 is l-asparagine, glutamine, Threonine, Serine or aspartic acid; The residue of corresponding X173 is aspartic acid or glycine; And the residue of corresponding X206 is methionine(Met) or glutamine.In some embodiments, except above feature, described structural domain or region can comprise that to be selected from following feature one or more further: the residue of corresponding X95 is α-amino-isovaleric acid or L-Ala; The residue of corresponding X97 is Methionin, arginine or leucine; The residue of corresponding X106 is L-glutamic acid or aspartic acid; The residue of corresponding X111 is leucine or methionine(Met); The residue of corresponding X126 is Isoleucine or α-amino-isovaleric acid; The residue of corresponding X127 is glutamine or arginine; The residue of corresponding X147 is phenylalanine, leucine or Serine; The residue of corresponding X152 is Threonine, Serine or methionine(Met); The residue of corresponding X163 is α-amino-isovaleric acid or Isoleucine; The residue of corresponding X173 is aspartic acid or glycine; The residue of corresponding X177 is Methionin or arginine; The residue of corresponding X192 is Methionin, arginine or L-glutamic acid; The residue of corresponding X194 is proline(Pro), glycine, aspartic acid, arginine or leucine; The residue of corresponding X198 is aspartic acid or glycine; The residue of corresponding X200 is proline(Pro), L-glutamic acid or Methionin; The residue of corresponding X208 is glutamine, Histidine or arginine; The residue of corresponding X210 is Threonine or L-Ala; And the residue of corresponding X211 is Methionin or L-glutamic acid.In some embodiments, with based on SEQ ID NO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.
In some embodiments, the ketoreductase improved comprise have corresponding based on SEQ ID NO:143,144 or 145 the region of residue 90-211 of list type or the aminoacid sequence of structural domain, it has at least following characteristics: the residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue polar residues, particularly Serine of corresponding X145; And the residue of corresponding X157 is polarity or acidic residues, particularly Serine or Threonine.In some embodiments, with based on SEQID NO:2,4 or 142 the counter structure territory of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein said aminoacid sequence with there is above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 90-211 of reference sequences, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprise have corresponding based on SEQ ID NO:143,144 or 145 the region of residue 90-211 of list type or the aminoacid sequence of structural domain, it has at least following characteristics: the residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue polar residues, particularly Serine of corresponding X145; And the residue of corresponding X157 is polarity or acidic residues, particularly Serine or Threonine.In some embodiments, with based on SEQ ID NO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein said aminoacid sequence with there is above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 90-211 of reference sequences, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprise have corresponding based on SEQ ID NO:143,144 or 145 the region of residue 90-211 of list type or the aminoacid sequence of structural domain, it has at least following characteristics: the residue of corresponding X94 is polarity, alkalescence, aliphatics or non-polar residue, particularly Threonine or α-amino-isovaleric acid; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue polar residues, particularly Serine of corresponding X145; And the residue of corresponding X157 is polarity or acidic residues, particularly Serine or Threonine.In some embodiments, with based on SEQ IDNO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein said aminoacid sequence with there is above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 90-211 of reference sequences, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprise have corresponding based on SEQ ID NO:143,144 or 145 the region of residue 90-211 of list type or the aminoacid sequence of structural domain, it has at least following characteristics: the residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues, particularly l-asparagine, proline(Pro), L-Ala or L-glutamic acid; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue polar residues, particularly Serine of corresponding X145; And the residue of corresponding X157 is polarity or acidic residues, particularly Serine or Threonine.In some embodiments,, the region of corresponding residue 90-211 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 sudden change extraly at other amino-acid residue places of structural domain.In some embodiments, with based on SEQ ID NO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have about 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein said aminoacid sequence with there is above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 90-211 of reference sequences, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprise have corresponding based on SEQ ID NO:143,144 or 145 the region of residue 90-211 of list type or the aminoacid sequence of structural domain, it has at least following characteristics: the residue of corresponding X94 is nonpolar or polar residues, particularly Threonine or α-amino-isovaleric acid; The residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues, particularly l-asparagine, proline(Pro), L-Ala or L-glutamic acid; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Serine or Threonine.In some embodiments, with based on SEQ ID NO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein said aminoacid sequence with there is above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 90-211 of reference sequences, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprise have corresponding based on SEQ ID NO:143,144 or 145 the region of residue 90-211 of list type or the aminoacid sequence of structural domain, it has at least following characteristics: the residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Serine or Threonine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, with based on SEQ ID NO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein said aminoacid sequence with there is above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 90-211 of reference sequences, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprise have corresponding based on SEQ ID NO:143,144 or 145 the region of residue 90-211 of list type or the aminoacid sequence of structural domain, it has at least following characteristics: the residue of corresponding X94 is nonpolar or polar residues, particularly Threonine or α-amino-isovaleric acid; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Serine or Threonine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, with based on SEQ ID NO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein said aminoacid sequence with there is above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 90-211 of reference sequences, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprise have corresponding based on SEQ ID NO:143,144 or 145 the region of residue 90-211 of list type or the aminoacid sequence of structural domain, it has at least following characteristics: the residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues, particularly l-asparagine, proline(Pro), L-Ala or L-glutamic acid; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Serine or Threonine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, with based on SEQ ID NO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein said aminoacid sequence with there is above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 90-211 of reference sequences, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprise have corresponding based on SEQ ID NO:143,144 or 145 the region of residue 90-211 of list type or the aminoacid sequence of structural domain, it has at least following characteristics: the residue of corresponding X94 is nonpolar or polar residues, particularly Threonine or α-amino-isovaleric acid; The residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues, particularly l-asparagine, proline(Pro), L-Ala or L-glutamic acid; The residue of corresponding X108 is alkalescence, restriction or aromatic moieties, particularly Histidine; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Serine or Threonine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, with based on SEQID NO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein said aminoacid sequence with there is above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 90-211 of reference sequences, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprise have corresponding based on SEQ ID NO:143,144 or 145 the region of residue 90-211 of list type or the aminoacid sequence of structural domain, it has at least following characteristics: the residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Serine or Threonine; The residue of corresponding X173 is acid or non-polar residue, particularly glycine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, with based on SEQ ID NO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein said aminoacid sequence with there is above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 90-211 of reference sequences, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprise have corresponding based on SEQ ID NO:143,144 or 145 the region of residue 90-211 of list type or the aminoacid sequence of structural domain, it has at least following characteristics: the residue of corresponding X94 is nonpolar or polar residues, particularly Threonine or α-amino-isovaleric acid; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Serine or Threonine; The residue of corresponding X173 is acid or non-polar residue, particularly glycine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, with based on SEQ ID NO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein said aminoacid sequence with there is above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 90-211 of reference sequences, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprise have corresponding based on SEQ ID NO:143,144 or 145 the region of residue 90-211 of list type or the aminoacid sequence of structural domain, it has at least following characteristics: the residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues, particularly l-asparagine, proline(Pro), L-Ala or L-glutamic acid; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Serine or Threonine; The residue of corresponding X173 is acid or non-polar residue, particularly glycine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, with based on SEQ ID NO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein said aminoacid sequence with there is above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 90-211 of reference sequences, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase improved comprise have corresponding based on SEQ ID NO:143,144 or 145 the region of residue 90-211 of list type or the aminoacid sequence of structural domain, it has at least following characteristics: the residue of corresponding X94 is nonpolar or polar residues, particularly Threonine or α-amino-isovaleric acid; The residue of corresponding X96 is restriction, aliphatics, nonpolar, acid or polar residues, particularly l-asparagine, proline(Pro), L-Ala or L-glutamic acid; The residue of corresponding X117 is nonpolar or polar residues, particularly Serine; The residue of corresponding X145 is polar residues, particularly Serine; And the residue of corresponding X157 is polarity or acidic residues, particularly Serine or Threonine; The residue of corresponding X173 is acid or non-polar residue, particularly glycine; And the residue of corresponding X206 is polarity or non-polar residue, particularly glutamine.In some embodiments, with based on SEQID NO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 90-211 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference in the quantity of the difference at other amino-acid residue places of structural domain.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprises the aminoacid sequence with at least above feature, and wherein said aminoacid sequence with there is above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 90-211 of reference sequences, there is the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase of improvement comprise have corresponding SEQ ID NO:143,144 or 145 the region of residue 1-89 of list type or the aminoacid sequence of structural domain.In some embodiments, the residue of the corresponding X7 in structural domain is polarity or restriction residue, particularly Serine, Histidine or l-asparagine.In some embodiments, with SEQ ID NO:2,4 or 142 reference sequences contrast, the region of corresponding residue 1-89 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places of structural domain.In some embodiments, in structural domain, the quantity of difference can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference.In some embodiments, contrast with reference sequences, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprise with there is at residue X7 place above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 1-89 of reference sequences, there is the aminoacid sequence of the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the region of corresponding residue 1-89 or structural domain can have that to be selected from following feature one or more: the residue of corresponding X3 is polarity, acidity or aromatic moieties, particularly tyrosine or aspartic acid; The residue of corresponding X7 is nonpolar, polarity or restriction residue, particularly Serine, Histidine or l-asparagine; The residue of corresponding X11 is aliphatics, nonpolar or polar residues, particularly Isoleucine or Threonine; The residue of corresponding X16 is aliphatics or non-polar residue, particularly Threonine, L-Ala, α-amino-isovaleric acid or glycine; The residue of corresponding X19 is nonpolar or aliphatic residue, particularly Isoleucine or α-amino-isovaleric acid; The residue of corresponding X23 is nonpolar or aromatic moieties, particularly phenylalanine; The residue of corresponding X41 is aliphatics, nonpolar or polar residues, particularly Serine, L-Ala or α-amino-isovaleric acid; The residue of corresponding X45 is aliphatics, nonpolar or polar residues, particularly L-glutamic acid or glycine; The residue of corresponding X49 is alkaline residue, particularly Methionin or arginine; The residue of corresponding X57 is aliphatics or non-polar residue, particularly Isoleucine or α-amino-isovaleric acid; The residue of corresponding X60 is aromatics, aliphatics, nonpolar or polar residues, particularly phenylalanine, α-amino-isovaleric acid or Threonine; The residue of corresponding X64 is aliphatics or non-polar residue, particularly L-Ala, Serine or Threonine; The residue of corresponding X72 is alkaline residue, particularly Methionin or arginine; And the residue of corresponding X82 is nonpolar or polar residues, particularly glycine or Serine.In some embodiments, with based on SEQ ID NO:2,4 or 142 reference sequences contrast, the region of corresponding residue 1-89 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-5,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places of structural domain.In some embodiments, in structural domain, the quantity of difference can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference.In some embodiments, contrast with reference sequences, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprise with there is at residue X7 place above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 1-89 of reference sequences, there is the aminoacid sequence of the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the ketoreductase of improvement comprise have corresponding SEQ ID NO:143,144 or 145 the region of residue 212-252 of list type or the aminoacid sequence of structural domain.In some embodiments, the residue of the corresponding X223 in structural domain is aliphatic residue, particularly α-amino-isovaleric acid.In some embodiments, with SEQ ID NO:2,4 or 142 reference sequences contrast, the region of corresponding residue 212-252 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places of structural domain.In some embodiments, in structural domain, the quantity of difference can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference.In some embodiments, contrast with reference sequences, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprise with there is at residue X223 place above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 212-252 of reference sequences, there is the aminoacid sequence of the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, the region of corresponding residue 212-252 or structural domain can have that to be selected from following feature one or more or at least whole: the residue of corresponding X214 is nonpolar, aliphatics or polar residues, particularly methionine(Met), α-amino-isovaleric acid, Threonine or Serine; The residue of corresponding X217 is aromatics, aliphatics or non-polar residue, particularly Isoleucine or phenylalanine; The residue of corresponding X223 is aliphatics or non-polar residue, particularly Isoleucine or α-amino-isovaleric acid; And the residue of corresponding X226 is nonpolar or aliphatic residue, particularly Isoleucine or α-amino-isovaleric acid.In some embodiments, with based on SEQ ID NO:2,4 or 142 the structural domain of reference sequences contrast, the region of corresponding residue 212-252 or structural domain can have 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18 or 1-20 residue difference extraly at other amino-acid residue places.In some embodiments, other residue places in structural domain, the quantity of difference can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18 or about 20 residue difference.In some embodiments, difference comprises conservative variants.In some embodiments, Ketoreductase polypeptides comprise with there is at residue X223 place above feature corresponding based on SEQ ID NO:2,4 or 142 the Amino acid sequences alignment of residue 212-252 of reference sequences, there is the aminoacid sequence of the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
In some embodiments, Ketoreductase polypeptides of the present disclosure can comprise with based on the reference sequences of SEQ IDNO:2, SEQ ID NO:4 or SEQ ID NO:142 or its region or structural domain, such as residue 90-211 has the aminoacid sequence of identity at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and condition is the residue of corresponding residue 145 is polar residues.In some embodiments, the residue of corresponding residue 145 can be polar residues, and polypeptide can be reduced into product at least about 70%s.e. by substrate.In some embodiments, the residue of corresponding residue 145 is Serines, and the one or more so that polypeptide had extraly in following replacement be better than further (in stereoselectivity, enzymatic activity and/or thermostability) wild-type Ke Feier ketoreductase or another engineering ketoreductase: 3 → Y (namely corresponding SEQ ID NO:2,4 or 142 the residue of residue 3 be replaced to tyrosine); 7 → S, N; 11 → T; 16 → A, V, G; 19 → V; 23 → F; 41 → V; 45 → G; 49 → R; 57 → V; 60 → T; 64 → T; 72 → R; 82 → S; 94 → T, R; 95 → A; 96 → P, A, E; 97 → R; 106 → D; 108 → H; 111 → M; 117 → S; 126 → V; 127 → R; 147 → L, S; 152 → M; 157 → T, S, D; 163 → I; 173 → G; 177 → R; 192 → R, E; 194 → G, D, N, L; 198 → G; 200 → K; 206 → Q; 208 → H, R; 210 → A; 211 → E; 214 → V, T; 217 → F; 223 → V; 226 → L; And 228 → A.
In some embodiments, the residue of corresponding residue 145 is Serines, and it is one or more to have in following replacement extraly, so that polypeptide is better than wild-type Ke Feier ketoreductase or another engineering ketoreductase further: 7 → S, N; 94 → T, V; 96 → P; 108 → H; 117 → S; 157 → T; 194 → N; 206 → Q; With 223 → V.
In some embodiments, the residue of corresponding residue 145 is Serines, and it is one or more to have in following replacement extraly, so that polypeptide is better than wild-type Ke Feier ketoreductase or another engineering ketoreductase further: 7 → S, N; 94 → T, V; 96 → P; 108 → H; 117 → S; 157 → T; 173 → G; 194 → N; 206 → Q; And 223 → V.
In some embodiments, each of the engineering ketoreductase of improvement described herein can comprise the disappearance of polypeptide described herein.Therefore, for each and each embodiment of Ketoreductase polypeptides of the present disclosure, disappearance can be 1 multiple amino acid of Ketoreductase polypeptides, 2 or more amino acid, 3 or more amino acid, 4 or more amino acid, 5 or more amino acid, 6 or more amino acid, 8 or more amino acid, 10 or more, 15 or more amino acid or 20 an or more amino acid, reach 10% of amino acid whose total quantity, reach 10% of amino acid whose total quantity, reach 20% of amino acid whose total quantity or reach 30% of amino acid whose total quantity, as long as keep the functionally active of ketoreductase activity.In some embodiments, disappearance can comprise 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10,1-11,1-12,1-14,1-15,1-16,1-18,1-20,1-22,1-24,1-25,1-30,1-35 or an about 1-40 amino acid.In some embodiments, disappearance can comprise the disappearance of 1-2,1-3,1-4,1-5,1-6,1-7,1-8,1-9,1-10 or 1-20 amino-acid residue.In some embodiments, the quantity of disappearance can be 1,2,3,4,5,6,7,8,9,10,11,12,14,15,16,18,20,22,24,26,30,35 or about 40 amino acid.In some embodiments, disappearance can comprise the disappearance of 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,18 or 20 amino-acid residue.
As skilled in the art to understand, unless otherwise indicated, some otherwise in the classification of amino-acid residue defined above are not mutually repel.Therefore, the amino acid with the side chain showing two or more physical-chemical properties can be included in multiple classification.The suitable classification of any amino acid or residue is obvious to those skilled in the art, particularly according to provided herein open in detail.
As described herein, Ketoreductase polypeptides of the present disclosure can exist with the form of fusion polypeptide, and wherein Ketoreductase polypeptides is blended in other polypeptide, such as antibody label (such as myc epi-position) or purification sequences (such as His label).Therefore, can use with other peptide fusion or the Ketoreductase polypeptides that do not merge.
In some embodiments, polypeptide described herein is not limited to the amino acid of genetic coding.Except the amino acid of genetic coding, polypeptide described herein completely or partially can comprise noncoding amino acid that is naturally occurring and/or synthesis.Some common undoded amino acid that polypeptide described herein can comprise includes but not limited to: the amino acid whose D-steric isomer of genetic coding; 2,3-diaminopropionic acid (Dpr); α-aminoacid (Aib); Epsilon-amino caproic acid (Aha); δ-aminovaleric acid (Ava); Sarcosine or sarkosine (MeGly or Sar); Ornithine (ORN); Citrulline (Cit); Tert-butylalanine (Bua); T-butylglycine (Bug); N-methyl isoleucine (MeIle); Phenylglycocoll (Phg); Cyclohexylalanine (Cha); Nor-leucine (Nle); Naphthylalanine (Nal); 2-chlorophenylalanine (Ocf); 3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf); 2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff); 4-fluorophenylalanine (Pff); 2-bromophenyl alanine (Obf); 3-bromophenyl alanine (Mbf); 4-bromophenyl alanine (Pbf); 2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf); 4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf); 3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf); 2-cyano group phenylalanine (Ocf); 3-cyano group phenylalanine (Mcf); 4-cyano group phenylalanine (Pcf); 2-trifluoromethyl-phenylalanine (Otf); 3-trifluoromethyl-phenylalanine (Mtf); 4-trifluoromethyl-phenylalanine (Ptf); 4-amino phenylalanine (Paf); 4-iodophenylalanine (Pif); 4-amino methyl phenylalanine (Pamf); 2,4 dichloro benzene L-Ala (Opef); 3,4-dichlorobenzene L-Ala (Mpcf); 2,4 difluorobenzene L-Ala (Opff); 3,4-difluorobenzene L-Ala (Mpff); Pyridine-2-base L-Ala (2pAla); Pyridin-3-yl L-Ala (3pAla); Pyridin-4-yl L-Ala (4pAla); Naphthalene-1-base L-Ala (1nAla); Naphthalene-2-base L-Ala (2nAla); Thiazolealanine (taAla); Benzothienyl L-Ala (bAla); Thienyl alanine (tAla); Furyl L-Ala (fAla); Hyperphenylalaninemia (hPhe); High tyrosine (hTyr); High tryptophan (hTrp); Penta fluoro benzene L-Ala (5ff); Styryl L-Ala (sAla); Anthryl L-Ala (aAla); 3,3-bis-phenylalanine (Dfa); 3-amino-5-phenyl valeric acid (Afp); Trolovol (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); β-2-thienyl alanine (Thi); Methionine sulfoxide (Mso); N (w)-Nitro-Arginine (nArg); High-lysine (hLys); (phosphonomethyl) phenylalanine (pmPhe); Phosphoserine (pSer); Phosphothreonine (pThr); High aspartic acid (hAsp); High glutamic acid (hGlu); 1-amino cyclopentyl-(2 or 3)-alkene-4 carboxylic acid; Pipecolic acid (PA), azetidine-3-carboxylic acid (ACA); 1-Aminocyclopentane-3-carboxylic acid; Allylglycine (aOly); PGIY (pgGly); High lactamine (hAla); Norvaline (nVal); Homoleucine (hLeu), high α-amino-isovaleric acid (hVal); Homoisoleucine (hIle); Homoarginine (hArg); N-acetyllysine (AcLys); 2,4-diamino-butanoic (Dbu); 2,3-DAB (Dab); N-methylvaline (MeVal); Homocysteine (hCys); Homoserine (hSer); Oxyproline (Hyp) and high proline(Pro) (hPro).The undoded amino acid that polypeptide described herein comprises other will be obvious (see such as Fasman to those skilled in the art, 1989, CRC Practical Handbook of Biochemistry andMolecular Biology (CRC biological chemistry and molecular biology practice handbook), CRC Press, Boca Raton, FL, each seed amino acid provided in 3-70 page and the reference wherein quoted, all described reference are incorporated to by reference).These amino acid can exist with L-configuration or D-form.
It will be apparent to those skilled in the art that amino acid containing protecting group side chain or residue also can comprise polypeptide described herein.When belonging to aromatics classification, this protected amino acid whose limiting examples comprises (protecting group is listed in bracket) but is not limited to: Arg (tos), Cys (methyl-benzyl), Cys (nitropyridine sulfeno), Glu (δ-benzyl ester), Gln (oxa-anthryl), Asn (N-δ-oxa-anthryl), His (bom), His (benzyl), His (tos), Lys (fmoc), Lys (tos), Ser (adjacent benzyl), Thr (adjacent benzyl) and Tyr (adjacent benzyl).
Polypeptide described herein can comprise, and the undoded amino acid of conformation restriction includes but not limited to N-methylamino acid (L-configuration); 1-amino cyclopentyl-(2 or 3)-alkene-4-carboxylic acid; Pipecolic acid; Azetidine-3-carboxylic acid; High proline(Pro) (hPro) and 1-Aminocyclopentane-3-carboxylic acid.
The polynucleotide of 7.3 coding engineering ketoreductases
In one aspect of the method, the disclosure provides the polynucleotide of coding engineering ketoreductase.Polynucleotide can be operatively attached to one or more heterologous regulatory sequence of controlling gene expression to produce the recombination of polynucleotide can expressing this polypeptide.The expression construct of the heterologous polynucleotide containing coding engineering ketoreductase can import suitable host cell to express corresponding Ketoreductase polypeptides.
Due to the understanding of codon corresponding to different aminoacids, the operability of protein sequence provides can the description of all polynucleotide of encoding target.The degeneracy (wherein same amino acid is encoded by optional or synonym) of genetic code allows the nucleic acid of preparation huge amount, the ketoreductase of their all improvement disclosed herein of all encoding.Therefore, after having authenticated specific aminoacid sequence, those skilled in the art by one or more codons of the mode modification sequence simply of the aminoacid sequence not change protein, can prepare any amount of different nucleic acid.In this respect, the disclosure covers the various of polynucleotide and each possible change especially, this is by selecting to select combination to prepare according to possible codon, and all these changes be considered to for any polypeptide institute disclosed herein especially disclosed in, the aminoacid sequence provided in table 2 is provided.
In different embodiments, preferably select codon to be applicable to wherein producing the host cell of this protein.Such as, for the preferred codon of bacterium for expressing the gene in bacterium; Be used for expressing in yeast for the preferred codon in yeast; And be used for expressing in mammalian cell for mammiferous preferred codon.Such as, the polynucleotide of SEQ ID NO:3 carry out codon optimized for expressing in intestinal bacteria (E.coli), but the ketoreductase of coding naturally occurring lactobacillus kefir in addition.
In some embodiments, polynucleotide comprise the nucleotide sequence of coding Ketoreductase polypeptides, described Ketoreductase polypeptides has and has at least about 85% with described herein with reference to any one in engineering Ketoreductase polypeptides, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, the aminoacid sequence of the sequence iden of 98% or 99% or larger, wherein said coding Ketoreductase polypeptides is included in corresponding SEQ ID NO:2, the residue place of the residue 145 of 4 or 142 has polar residues, the particularly aminoacid sequence of Serine.In some embodiments, polynucleotide encoding comprise be selected from SEQ ID NO:6,8,10,12,14,16,18,20,22,24,26,30,32,38,40,42,44,46,48,50,52,54,56,58,60,64,66,68,70,74,76,78,80,86,88,90,92,104,106,110,112,124,126, the engineering Ketoreductase polypeptides of the aminoacid sequence of 130 or 134.
In some embodiments, the polynucleotide of coding engineering ketoreductase are selected from SEQ ID NO:5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131 and 133.
In some embodiments, polynucleotide can hybridize one of the polynucleotide in the sequence comprising below correspondence under high stringency: SEQ ID NO:5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133, the substrate conversion of structural formula (I) can be become the function ketoreductase of the product of structural formula (II) by the polynucleotide encoding of wherein hybridizing under high stringency.
In some embodiments, polynucleotide comprise the nucleotide sequence of polypeptide described herein of encoding, but with the reference polynucleotide of coding engineering ketoreductase, there is on nucleotide level the sequence iden of about 80% or larger, the sequence iden of about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or larger, wherein the substrate conversion of structural formula (I) can be become the function ketoreductase of the product of structural formula (II) by polynucleotide encoding.In some embodiments, with reference to polynucleotide be selected from corresponding SEQ ID NO:5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129, the sequence of 131 and 133.
The polynucleotide of the separation of the Ketoreductase polypeptides that coding improves can process the expression providing polypeptide in many ways.According to expression vector, before the polynucleotide insertion vector that will be separated, may be needs or required to its process.The technology utilizing recombinant DNA method to modify polynucleotide and nucleotide sequence is known in the art.Guidance is provided in the people such as Sambrook, 2001, MolecularCloning:A Laboratory Manual (molecular cloning: laboratory manual), the 3rd edition, Cold SpringHarbor Laboratory Press; Compile with Current Protocols in Molecular Biology (up-to-date experimental methods of molecular biology compilation), Ausubel.F, Greene Pub.Associates, 1998, be updated to 2006.
For bacterial host cell, the suitable promoter of transcribing being used to guide nucleic acid construct of the present disclosure comprises from the following promotor obtained: E. coli lac operon, streptomyces coelicolor (Streptomyces coelicolor) agarase gene (dagA), subtilis (Bacillussubtilis) type froctosan saccharase gene (sacB), Bacillus licheniformis (Bacillus licheniformis) alpha-amylase gene (amyL), bacstearothermophilus (Bacillus stearothermophilus) maltogenic amylase gene (amyM), bacillus amyloliquefaciens (Bacillus amyloliquefaciens) alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), subtilis xylA and xylB gene and the protokaryon β-lactamase gene (people such as Villa-Kamaroff, 1978, Proc.NatlAcad.Sci.USA 75:3727-3731), and the tac promotor (people such as DeBoer, 1983, Proc.NatlAcad.Sci.USA 80:21-25).In addition, promotor is described in " Usefulproteins from recombinant bacteria (the useful proteins matter from recombinant bacteria) ", ScientiffcAmerican, 1980,242:74-94; And the people such as Sambrook, on seeing.
For filamentous fungal host cell, the suitable promoter of transcribing being used to guide nucleic acid construct of the present disclosure comprises the promotor obtained from following gene: aspergillus oryzae (Aspergillus oryzae) TAKA amylase, rhizomucor miehei (Rhizomucor miehei) aspartate protease, aspergillus niger (Aspergillus niger) neutral alpha-amylase, niger acid stable type α-amylase, aspergillus niger or Aspergillus awamori (Aspergillus awamori) glucoamylase (glaA), rhizomucor miehei lipase, line protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans (Aspergillusnidulans) acetamidase and Fusarium oxysporum (Fusarium oxysporum) trypsin like proteases (WO 96/00787), and NA2-tpi promotor (heterozygote from the promotor of Aspergillus ni ger neutral α-amylase and Aspergillus oryzae triose phosphate isomerase gene) and mutant thereof, brachymemma and hybrid promoter.
In yeast host, useful promotor can from yeast saccharomyces cerevisiae (Saccharomycescerevisiae) Hydratase, phosphoenolpyruvate (ENO-1), yeast saccharomyces cerevisiae galactokinase (GAL1), yeast saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP) and the kinase whose gene of yeast saccharomyces cerevisiae 3-phoshoglyceric acid.Other useful promotors for yeast host cell are described in the people such as Romanos, 1992, Yeast 8:423-488.
Control sequence can also be suitable transcription terminator sequences---a kind of sequence of being transcribed with termination by host cell identification.Terminator sequence is operably connected to 3 ' end of the nucleotide sequence of coded polypeptide.Any terminator worked in the host cell selected can be used for the present invention.
Such as, the exemplary transcription terminator for filamentous fungal host cell can obtain from the gene of oryzae TAKA amylase, aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, aspergillus niger alpha-glucosidase and Fusarium oxysporum trypsin enzyme sample proteolytic enzyme.
Exemplary terminator for yeast host cell can obtain from the gene of yeast saccharomyces cerevisiae Hydratase, phosphoenolpyruvate, S. cerevisiae cytochrome C (CYC1) and S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase.Other useful terminators for yeast host cell are described in the people such as Romanos, 1992, the same.
Control sequence can also be suitable leader sequence---the non-translational region of a kind of mRNA important to the translation of host cell.This leader sequence is operably connected to 5 ' end of the nucleotide sequence of coded polypeptide.Any leader sequence worked can be used in the host cell of selection.Exemplary leader for filamentous fungal host cell obtains from the gene of oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase.Suitable leader for yeast host cell obtains from the gene of yeast saccharomyces cerevisiae Hydratase, phosphoenolpyruvate (ENO-1), yeast saccharomyces cerevisiae glycerol 3-phosphate acid kinase, cerevisiae alpha-factor and yeast saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
Control sequence can also be polyadenylation se-quence---a kind of be operably connected to nucleotide sequence 3 ' end and be identified as the sequence of the signal adding poly-adenosine residue to the mRNA transcribed by host cell when transcribing.Any polyadenylation se-quence worked in the host cell selected can be used for the present invention.Exemplary polyadenylation se-quence for filamentous fungal host cell can from the gene of oryzae TAKA amylase, aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin enzyme sample proteolytic enzyme and aspergillus niger alpha-glucosidase.The polyadenylation se-quence useful to yeast host cell is described in Guo and Sherman, 1995, Mol Cell Bio15:5983-5990.
Control sequence can also be signal peptide coding region, and its coding is connected to the aminoterminal aminoacid sequence of polypeptide, and instructs coded polypeptide to enter emiocytosis approach.5 ' end of the encoding sequence of nucleotide sequence itself can contain by the natural signal peptide coding region being connected to the section of the coding region of the polypeptide of coding secretion of translation reading frame.Alternatively, 5 ' end of encoding sequence can containing the signal peptide coding region coming from encoding sequence outward.If encoding sequence is not natural in signal peptide coding region, then may need exogenous signals peptide-coding region.
Alternatively, exogenous signals peptide-coding region can replace natural signals peptide-coding region simply, thus strengthens the secretion of polypeptide.But any signal peptide coding region instructing the polypeptide of expressing to enter the Secretory Pathway of the host cell of selection can be used for the present invention.
Effective signal peptide coding region for bacterial host cell is the signal peptide coding region obtained from following gene: genus bacillus (Bacillus) NClB 11837 maltogenic amylase, bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, stearothermophilus neutral proteolytic enzyme (nprT, nprS, nprM) and subtilis prsA.Other signal peptide is described in Simonen and Palva, 1993, MicrobiolRev 57:109-137.
Effective signal peptide coding region for filamentous fungal host cell can be the signal peptide coding region obtained from following gene: oryzae TAKA amylase, Aspergillus ni ger neutral amylase, aspergillus niger glucoamylase, rhizomucor miehei aspartic protease, Humicola insolens (Humicola insolens) cellulase and Humicola lanuginosa (Humicola lanuginosa) lipase.
Can from the gene of cerevisiae alpha-factor and Saccharomyces cerevisiae invertase to the useful signal peptide of yeast host cell.Other useful signal peptide coding regions are described in the people such as Romanos, 1992, the same.
Control sequence can also be propeptide coding region, and its coding is positioned at the aminoterminal aminoacid sequence of polypeptide.The polypeptide produced is called proenzyme (proenzyme) or propolypeptide (or being called proenzyme (zymogen) in some cases).The usual non-activity of propolypeptide, and by being converted into ripe active polypeptide from propolypeptide catalysis or autocatalysis cutting propetide.Propeptide coding region can obtain from following gene: bacillus subtilis alkali proteinase (aprE), Bacillus subtilis neutral proteolytic enzyme (nprT), cerevisiae alpha-factor, rhizomucor miehei aspartic protease and thermophilic fungus destroyed wire (Myceliophthora thermophila) Sumylact L (WO 95/33836).
If signal peptide and propetide district are all present in the N-terminal of polypeptide, then propetide district is positioned at the consecutive position of amino terminus, and signal peptide district is positioned at the aminoterminal consecutive position in propetide district.
Also can expect to add and regulate sequence, it allows relative host cell growth to regulate the expression of polypeptide.The example of regulation system is the regulation system causing response chemistry or the physical stimulation existence of compound (comprise regulate) and open or close the expression of gene.In prokaryotic host cell, suitable adjustment sequence comprises lac, tac and trp operator gene system.In yeast host cell, suitable regulation system comprises, such as ADH2 system or GAL1 system.In filamentous fungus, suitable adjustment sequence comprises TAKA α-amylase promotor, aspergillus niger glucoamylase promotor and aspergillus oryzae glucoamylase promotor.
Other examples of sequence are regulated to be the sequences allowing gene amplification.In eukaryotic system, these are included in the dihydrofolate reductase gene of amplification when methotrexate exists and the metallothionein gene with heavy metal amplification.In these cases, encode KRED polypeptide of the present invention nucleotide sequence will with adjustment sequence be operably connected.
Therefore, in some embodiments, the disclosure also relates to recombinant expression vector, according to the type of the host that they import, it comprises the polynucleotide of coding engineering Ketoreductase polypeptides or its variant, and one or more Expression modulation district (such as promotor and terminator), replication orgin etc.Various nucleic acid described above and control sequence can connect together to produce recombinant expression vector, and it can comprise one or more restriction site easily and insert or replacement in this site to make the nucleotide sequence of coded polypeptide.Alternatively, by nucleotide sequence or the nucleic acid construct that comprises this sequence being inserted into suitable carrier for expressing to express nucleotide sequence of the present disclosure.In generation expression vector, encoding sequence is arranged in carrier, is operably connected to appropriate control sequences for expressing to make encoding sequence.
Recombinant expression vector can be can stand recombinant DNA operation easily and any carrier (such as plasmid or virus) that polynucleotide sequence can be made to express.The selection of carrier will depend on the consistency of the host cell that carrier and this carrier import usually.Carrier can be straight line or closed circular plasmid.
In some embodiments, expression vector can be autonomously replicationg vector, and namely as the carrier that extrachromosomal entity exists, it copies independent of chromosome duplication, and this carrier is plasmid, extra-chromosomal element, minichromosome or artificial chromosome such as.This carrier can comprise any parts (means) guaranteeing self-replication.Alternatively, this carrier can be integrated in genome and the carrier copied together with its karyomit(e) integrated when importing host cell.In addition, single carrier or plasmid can be used, or together containing two or more carriers of genomic STb gene or the plasmid that are directed to host cell, or transposon.
Expression vector of the present disclosure can contain one or more selection marker thing, and it allows to be easy to select transformant.Selection marker thing is a kind of gene, and its product provides biocide or virus resistance, the resistance of heavy metal, auxotrophic prototroph, and similarity.The example of bacterium selection marker thing is the dal gene from subtilis or Bacillus licheniformis, or gives antibiotics resistance, the such as mark of Ampicillin Trihydrate, kantlex, paraxin (embodiment 1) or tetracyclin resistance.The mark being suitable for yeast host cell is ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3.
The selection marker thing used in filamentous fungal host cell includes but not limited to amdS (acetamidase), argB (ornithine carbamyl transferase), bar (phosphinothricin acetyl transferase), hph (hygromix phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenylyl transferase) and trpC (anthranilate synthase), and its equivalent.The embodiment used in aspergillus cell comprises amdS and the pyrG gene of Aspergillus nidulans or aspergillus oryzae, and the bar gene of streptomyces hygroscopicus (Streptomyces hygroscopicus).
Expression vector of the present invention preferably comprise allow this vector integration to host cell genome or allow carrier in cell independent of the element of genome self-replicating.For being incorporated into host cell gene group, this carrier can rely on the nucleotide sequence of coded polypeptide or any other element of carrier, thus by homology or non-homogeneous restructuring by vector integration to genome.
Alternatively, expression vector can comprise the genomic other nucleotide sequence being used to guide and being incorporated into host cell by homologous recombination.This other nucleotide sequence can make carrier be incorporated into host cell gene group in chromosomal exact position.In order to be increased in the possibility that exact position is integrated, this integrated element preferably should comprise sufficient amount with the nucleic acid of corresponding target sequence very high homology, such as 100 to 10,000 base pair, preferably 400 to 10,000 base pair and most preferably 800 to 10,000 base pair are to increase the possibility of homologous recombination.Integrated element can be any sequence with the target sequence homology in the genome of host cell.In addition, integrated element can be the nucleotide sequence of non-coding or coding.On the other hand, carrier is by the genome of non-homologous re-combination to host cell.
For self-replicating, carrier can comprise the replication orgin that can make carrier self-replicating in relevant host cell further.The example of bacterial origin of replication is P15A ori, or the pBR322 plasmid allowing to copy in intestinal bacteria, pUC19, pACYC177 (this plasmid has P15A ori) or pACYC184 replication orgin, and allow the replication orgin of pUB110, pE194, pTA1060 or pAM β 1 copied in genus bacillus.The example of the replication orgin used in yeast host cell is replication orgin ARS1, ARS4 of 2 microns, the combination of ARS1 and CEN3 and the combination of ARS4 and CEN6.Replication orgin can be the replication orgin (see such as Ehrlich, 1978, Proc Natl Acad Sci.USA75:1433) with the temperature sensitive sudden change of the function made it in host cell.
The more than one copy of nucleotide sequence of the present invention can be inserted into host cell to increase the production of gene product.By at least one other copy of sequence is incorporated in host cell gene group, or the increase of the number of copies of nucleotide sequence is obtained by comprising the selection marker thing gene (if cell comprises the amplification copy of selection marker thing gene, then the other copy of nucleotide sequence is by screening there is culturing cell under suitable selective agent) that can increase with nucleotide sequence.
Commercially available for many in the expression vector in the present invention.Suitable business expression vector comprises from Sigma-Aldrich Chemicals, the p3xFLAGTMTM expression vector of St.Louis MO., it comprises CMV promoter for expressing in mammalian host cell and hGH site of polyadenylation, and for the pBR322 replication orgin that increases in intestinal bacteria and Ampicillin resistant marker gene.Other suitable expression vectors are can from Stratagene, the pBluescriptII SK (-) that LaJolla CA business obtains and pBK-CMV, and from the derivative plasmid of pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or the pPoly (people such as Lathe, 1987, Gene 57:193-201).
7.4 for expressing the host cell of Ketoreductase polypeptides
In one aspect of the method, disclosure providing package is containing the host cell of the polynucleotide of the disclosed Ketoreductase polypeptides improved of code book, and these polynucleotide are operably connected in host cell for expressing one or more control sequences of ketoreductase.Known in the art for expressing by the host cell of the KRED polypeptide of expression vector codes of the present invention, and include but not limited to bacterial cell, such as intestinal bacteria, lactobacillus kefir, short lactobacillus, streptomycete (Streptomyces) and Salmonella typhimurium (Salmonella typhimurium) cell; Fungal cell, such as yeast cell (such as yeast saccharomyces cerevisiae or pichia spp (Pichia pastoris) (ATCC accession number 201178)); Insect cell, such as fruit bat S2 and spodoptera Sf9 (Spodoptera Sf9) cell; Zooblast, such as CHO, COS, BHK, 293 and Bowes melanoma cell; And vegetable cell.The suitable substratum of host cell described above and growth conditions are known in the art.
The polynucleotide transfered cell of expressing ketoreductase can will be used for by various methods known in the art.Technology especially comprises electroporation, particle gun particle bombardment, liposome-mediated transfection, calcium chloride transfection and protoplast fusion.For will be obvious by the various methods of polynucleotide transfered cell to those skilled in the art.
Exemplary host cells is intestinal bacteria (Escherichia coli) W3110.Be operably connected to plasmid pCK110900 by the polynucleotide of ketoreductase improved by coding, and then be operably connected to the lac promotor that controlled by lacI repressor to produce expression vector.Expression vector also comprises P15a replication orgin and chloramphenicol resistance gene.Cell containing tested polynucleotide in intestinal bacteria W3110 is selected to be separated by making cell stand paraxin.
The method of 7.5 generation engineering Ketoreductase polypeptides
In some embodiments, in order to prepare KRED polynucleotide and the polypeptide of improvement of the present disclosure, obtain the naturally occurring ketoreductase of (or derivative) catalytic reduction reaction from lactobacillus kefir or short lactobacillus.In some embodiments, parent polynucleotide sequence is carried out codon optimized with the expression strengthening ketoreductase in particular host cell.Exemplarily, the parent polynucleotide sequence of the KRED polypeptide of encoding wild type lactobacillus kefir is built from the oligonucleotide prepared according to the known peptide sequence (Genbank accession number AAP94029GI:33112056) of lactobacillus kefir KRED sequence obtainable Genbank database.By codon optimized for parent polynucleotide sequence with at expression in escherichia coli, and codon optimized polynucleotide are cloned in expression vector, under the control expression of ketoreductase gene being placed in lac promotor and lacI repressor gene.Differentiate the clone at the active ketoreductase of expression in escherichia coli, and sequenced genes is to determine their identity.The sequence (SEQ ID NO:1) of specifying is the parental sequences being used as the major part experiment of the engineering ketoreductase of evolving from lactobacillus kefir ketoreductase and the starting point of library construction.
As previously mentioned, by making the polynucleotide of the naturally occurring ketoreductase of coding stand mutagenesis and/or directed evolution method can obtain engineering ketoreductase.Exemplary orientation evolution technology is mutagenesis and/or DNA shuffling, as described in the following: Stemmer, and 1994, Proc Natl Acad Sci USA91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and United States Patent (USP) 6,537,746.Inter alia, available other orthogenesiss operation comprises staggered extension process (StEP), the vitro recombination (people such as Zhao, 1998, Nat.Biotechnol.16:258-261), the mutagenesis PCR (people such as Caldwell, 1994, and cassette mutagenesis (people such as Black, 1996, Proc NatlAcadSci USA 93:3525-3529) PCRMethodsAppl.3:S136-S140).
The clone obtained after mutagenic treatment, screening has the engineering ketoreductase of the required enzymatic property improved.Because NADH or NADPH is converted into NAD +or NADP +, therefore use the standard biochemical techniques of the minimizing speed of monitoring NADH or NADPH concentration (minimizing via absorbancy or fluorescence) can to measure the enzymic activity from expression library.(for example, see embodiment 7) in this reaction, when ketone substrate to be reduced to corresponding hydroxyl by ketoreductase, ketoreductase consumption (oxidation) NADH or NADPH.As the minimizing by absorbancy or fluorescence surveyed, the minimizing speed of time per unit NADH or NADPH concentration shows that relative (enzymatic) of KRED polypeptide in the lysate (or the lyophilized powder prepared thus) of fixed amount is active.If the enzymatic property of required improvement is thermostability, then can stand at enzyme preparation the temperature determined and after measuring thermal treatment remaining enzymic activity amount measured enzymic activity afterwards.Then be separated the clone of polynucleotide containing coding ketoreductase, and to its order-checking with the change (if there is) differentiating nucleotide sequence, and use it for express enzyme in host cell.
According to known synthetic method, if know the sequence of engineered polypeptide, then the polynucleotide of codase are prepared by Standard solid-phase techniques.In some embodiments, the fragment up to about 100 bases can be synthesized individually, then connect (such as by enzymatic or chemical connection process, or polymerase-mediated method) to form any required continuous sequence.Such as, polynucleotide of the present invention and oligonucleotide are prepared by chemosynthesis, it uses the people such as such as Beaucage, the classical phosphoamidite method described in 1981, TetLett22:1859-69, or the people such as Matthes, 1984, EMBO J.3:801-05 in describe method, such as, as its usually in automatic synthesis method put into practice.According to phosphoamidite method, oligonucleotide is synthesized (such as in automatic dna synthesizer), purifying, annealing, connection being cloned in suitable carrier.In addition, substantially any nucleic acid any one can obtain from various commercial source, described commercial source is The Midland Certified ReagentCompany such as, Midland, TX, The Great American Gene Company, Ramona, CA, ExpressGen Inc.Chicago, IL, Operon Technologies Inc., Alameda, CA and other companies many.
The engineering ketoreductase of expressing in host cell can reclaim from cell and/or substratum, it use any one or multiple in protein purification known technology, especially comprise N,O-Diacetylmuramidase process, ultrasonic, filter, saltout, ultracentrifugation and chromatogram.Appropriate solution for dissolving and high efficiency extraction protein from bacterium (such as intestinal bacteria) is the trade name CelLytic B with the Sigma-Aldrich of St.Louis MO tMcommercially available.
Chromatographic technique for separating of Ketoreductase polypeptides especially comprises reverse-phase chromatography high performance liquid chromatography, ion-exchange chromatography, gel electrophoresis and affinity chromatography.Condition for purifying certain enzyme will partly depend on the factors such as such as net charge, hydrophobicity, wetting ability, molecular weight, shape of molecule, and will be obvious to those skilled in the art.
In some embodiments, affine technology can be used for being separated the ketoreductase improved.For affinity chromatography purifying, any antibody of specific binding Ketoreductase polypeptides can be used.In order to produce antibody, various host animal, includes but not limited to rabbit, mouse, rat etc., by coming immune with polypeptide injection.This polypeptide is connected to suitable carrier, such as BSA by side chain functionalities or the linker that is connected to side chain functionalities.According to host species, various adjuvant can be used to increase immunne response, the human adjuvants that adjuvant includes but not limited to freund's adjuvant (completely with incomplete), mineral coagulant (such as aluminium hydroxide), surfactant (such as lysolecithin), Pluronic Polyols (pluronic polyo1), polyanion, peptide, oil-emulsion, keyhole limpet hemocyanin, dinitrophenol(DNP) and comes in handy, such as BCG (bacille Calmette-Guerin vaccine) and CBP (Corynebacterium parvum).
The method of 7.6 use engineering ketoreductases and compound prepared therefrom
Ketoreductase described herein can ketone group in the compound 3-ketone group tetramethylene sulfide of catalytic structure formula (I)
To the reduction reaction of chiral alcohol product (the R)-3-hydroxy tetrahydro thiophene of counter structure formula (II)
In some embodiments, the disclosure provides the method for the product substrate of the compound of structural formula (I) being reduced into structural formula (II), and wherein said method comprises and is being suitable for contacting under reaction conditions substrate being reduced into alcohol product or hatching substrate with Ketoreductase polypeptides of the present disclosure.
Such as, in some embodiments of present method, respectively with SEQ ID NO:4,2 and 142 wild-type lactobacillus kefir or short lactobacillus or Lactobacillus minor KRED alignment, the residue place that Ketoreductase polypeptides is included in corresponding X145 has polar residues, particularly the aminoacid sequence of Serine.As referred to herein, with SEQ ID NO:2, the reference sequences contrast of 4 or 106, Ketoreductase polypeptides also can have one or more sudden change at other amino-acid residue places.These differences are described in SEQID NO:143, the list type of 144 and 145 and description herein.
In some embodiments of this method, Ketoreductase polypeptides comprises, such as and be not limited to, with the residue place at corresponding X145 have polar residues particularly Serine based on SEQ ID NO:2,4 or 142 reference sequences there is the aminoacid sequence of the identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, wherein Ketoreductase polypeptides also has polar residues at the residue place of corresponding X145, particularly Serine.
In some embodiments of present method, product is reduced so that the steric isomer being greater than about 65% is excessive, wherein Ketoreductase polypeptides comprise corresponding SEQ ID NO:6,8,10,12,14,16,18,20,22,24,26,30,32,38,40,42,44,46,48,50,52,54,56,58,60,64,66,68,70,74,76,78,80,86,88,90,92,104,106,110,112,124,126, the sequence of 130 or 134.
In some embodiments of present method, product is reduced into product so that the steric isomer being greater than about 90% is excessive, wherein Ketoreductase polypeptides comprises corresponding SEQ ID NO:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106, 108, 110, 112, 126, 128, the aminoacid sequence of 130 or 134.
In some embodiments of present method, product is reduced into product to be greater than about 98%s.e, wherein Ketoreductase polypeptides comprise corresponding SEQ ID NO:6,8,10,18,20,22,24,26,28,30,34,36,38,40,42,50,52,54,58,62,66,70,72,76,78, the aminoacid sequence of 80 and 134.
In some embodiments of the method for substrate being reduced into product, substrate is reduced into product with the speed of the raising contrasted with wild-type enzyme (SEQ ID NO:4), wherein said Ketoreductase polypeptides comprise corresponding SEQ ID NO:6,8,10,12,14,16,18,20,22,24,26,30,32,38,40,42,44,46,48,50,52,54,56,58,60,64,66,68,70,74,76,78,80,86,88,90,92,104,106,110,112,124,126, the aminoacid sequence of 130 or 134.
In some embodiments, reaction conditions is pH 7.5 or lower.In some embodiments, reaction conditions is the pH from about 5.0 to about 7.5.In some embodiments, reaction conditions is the pH from about pH 6.0 to about 7.5.In some embodiments, reaction conditions temperature is about 25 DEG C or lower.In some embodiments, reaction conditions is 5 DEG C to about 25 DEG C, or the temperature of 5 DEG C to 15 DEG C.In some embodiments, reaction conditions also comprises cofactor regeneration system, as described further below.
In some embodiments, method for substrate being reduced into product can comprise and contacts substrate with ketoreductase of the present disclosure comprising at least 100g/L substrate and the reaction conditions of at least 0.8-1.0g/L Ketoreductase polypeptides, and wherein the substrate of at least 90% changes into product in (such as 20-24 hour) being less than 24 hours.In some embodiments, the substrate being greater than 95%, 96%, 97%, 98% or 99% or more changes into product at reaction conditions.Exemplary polypeptide for the method include but not limited to comprise corresponding SEQ ID NO:6,8,10,12,14,18,20,22,24,26,30,32,38,40,42,44,46,48,50,52,54,56,58,60,64,66,68,70,74,76,78,80,82,86,88,90,92,104,106,110,112,126, the polypeptide of the aminoacid sequence of 130 and 134.
In some embodiments, method for substrate being reduced into product can comprise and contacts substrate with ketoreductase of the present disclosure comprising at least 100g/L substrate and the reaction conditions of at least 0.8-1.0g/L Ketoreductase polypeptides, and wherein the substrate of at least 90% changed into product in 12-20 hour.In some embodiments, the substrate being greater than 95%, 96%, 97%, 98% or 99% or more changes into product at reaction conditions.Exemplary polypeptide for the method includes but not limited to SEQ ID NO:26,44,68 and 104.
In some embodiments, any one in Ketoreductase polypeptides provided herein can be used for producing (R)-3-hydroxy tetrahydro thiophene, and it is for the production of multi-medicament, the intermediate of such as microbiotic and proteinase inhibitor.In some embodiments, Ketoreductase polypeptides can be used for the microbiotic sulopenem (CP-70,429) that production has following structural formula (III), and salt, and solvate and hydrate:
Therefore, in some embodiments, at microbiotic (the i.e. CP-70 for the synthesis of structural formula (III), 429) in method, step in method can comprise and being contacted with any one in ketoreductase described herein by the substrate of formula (I), thus by substrate conversion or the product being reduced into structural formula (II).
The product of structural formula (II) is by J.Org.Chem., and the route of synthesis described in 1992,57:4352 changes into the sulopenem with structural formula (III), and intermediate, and described document is incorporated at this by reference.The approach wherein described is described in hereafter, and wherein Ts is p-toluenesulfonyl, and Ac is ethanoyl, and TBS is t-butyldimethylsilyi.
In some embodiments, method provides the synthesis of each of intermediate of structural formula (IV), (V), (VI), (VII), (VIII), (IX), (X) and (XI), the step in the method for wherein synthetic intermediate can be included in be applicable to by substrate conversion or be reduced into structural formula (II) product condition under with the substrate of any one contact (I) in ketoreductase described herein.
Other one or more steps in this method can according to J.Org.Chem., and describe operation or its improvement of one or more step in 1992,57:4352, described document is incorporated to by reference.In order to the product conversion of structural formula (II) being become the intermediate of structural formula (IV), at J.Org.Chem., the dimethyl aminopyridine reagent used in the operation that 1992,57:4352 describes can replace with pyridine or 1-Methylimidazole.In order to the intermediate of the converted one-tenth structure formula V by structural formula (IV), at J.Org.Chem., 1992, the reaction carried out at 0 DEG C in the operation that 57:4352 describes can in lower temperature, such as, carry out at-25 DEG C, minimize to make the formation of corresponding sulfone by product further.
In some embodiments, (R)-3-hydroxy tetrahydro thiophene intermediate can be used for synthesis HIV-proteinase inhibitor (J.Med.Chem., 1994,37:1177) or other compounds or medicine, their synthesis depends on intermediate (R)-3-hydroxy tetrahydro thiophene (formula (II)), wherein comprises for the synthesis of the step in the method for medical compounds and uses any one in Ketoreductase polypeptides provided herein by the reduction of the compound of formula (I) or the compound transforming an accepted way of doing sth (II).
As is known to the person skilled in the art, the enzymatic reduction reaction of ketone body powder needs cofactor usually.Usually also cofactor is needed, although the cofactor that needs of many embodiments of engineering ketoreductase is far fewer than with the enzymatic reaction of wild-type ketone body powder by the enzymatic reduction reaction of engineering ketone body powder described herein.As used herein, term " cofactor " refers to the non-protein compound with ketoreductase combinations.The cofactor being applicable to using together with engineering ketoreductase described herein includes but not limited to NADP +(Triphosphopyridine nucleotide, reduced), NADPH (NADP +reduction form), NAD +(Reduced nicotinamide-adenine dinucleotide) and NADH (NAD +reduction form).In general, the cofactor of reduction form is added to reaction mixture.Reduced-NAD (P) H-shaped formula can use cofactor regeneration system from oxidized form NAD (P) +form optionally regenerates.
Term " cofactor regeneration system" refer to reaction (the such as NADP of the cofactor participating in reduction-oxidation form +to NADPH) a group reaction thing.The cofactor be oxidized by the reduction of the enzymatic ketone substrate of ketone body powder is regenerated as reduction form by cofactor regeneration system.Cofactor regeneration system comprises stoichiometric reductive agent, and it is the source going back protohydrogen equivalent, and can the cofactor of reduction-oxidation form.Cofactor regeneration system can comprise catalyzer further, such as enzyme catalyst, the cofactor of its catalytic reducer reduction-oxidation form.From NAD +or NADP +the cofactor regeneration system of regeneration of NAD H or NADPH is known in the art respectively, and can be used in method described herein.
Spendable suitable exemplary cofactor regeneration system includes but not limited to glucose and glucose dehydrogenase, formate and hydrogenlyase, glucose 6-phosphoric acid and glucose-6-phosphate dehydrogenase, secondary alcohol (such as Virahol) and dehydrogenating para-alcohol enzyme, phosphorous acid ester and phosphorous acid desaturase, molecular hydrogen and hydrogenase, and similar system.These systems can with the NADP as cofactor +/ NADPH or NAD +/ NADH combinationally uses.The electrochemical regeneration of hydrogenase is used also to can be used as cofactor regeneration system.See, such as United States Patent (USP) the 5th, 538,867 and 6,495, No. 023, be incorporated at this by reference both them.The chemical cofactor regeneration system comprising metal catalyst and reductive agent (such as, molecular hydrogen or formate) is also suitable.Announce WO 2000/053731 see such as PCT, it is incorporated at this by reference.
Herein, term " glucose dehydrogenase " and " GDH " are used interchangeably to refer to NAD +or NADP +dependent enzyme, they are catalysis D-Glucose and NAD respectively +or NADP +be converted into glyconic acid and NADH or NADPH.Following reaction formula (1) describes the glucose of glucose dehydrogenase enzyme catalysis to NAD +or NADP +reduction.
The glucose dehydrogenase being suitable for the practice of method described herein comprises the glucose dehydrogenase of naturally occurring glucose dehydrogenase and non-natural existence.Naturally occurring glucose dehydrogenase enzyme coding gene is reported in the literature.Such as subtilis 61297GDH gene is expressed in intestinal bacteria, and report that it shows the identical physicochemical property (people such as Vasantha of the enzyme that produces in host intrinsic with it, 1983, Proc.Natl.Acad.Sci.USA 80:785).Corresponding to the gene order of the subtilis GDH gene of Genbank accession number M12276 by people such as Lampel, 1986, J.Bacteriol.166:238-243 reports, and by people such as Yamane, 1996, Microbiology 142:3047-3056 reports that its correction form is Genbank accession number D50453.Naturally occurring GDH gene also comprises coding from bacillus cereus (B.cereus) ATCC14579 (Nature, 2003,423:87-91; Genbank accession number AE017013) and bacillus megaterium (B.megaterium) (Eur.J.Biochem., 1988,174:485-490, Genbank accession number X12370; J.Ferment.Bioeng., 1990,70:363-369, Genbank accession number GI216270) the gene of GDH.Glucose dehydrogenase from genus bacillus (Bacillus sp.) is provided as SEQ ID NO:10 and 12 (respectively by the polynucleotide sequence coding corresponding to the SEQ ID NO:9 and 11 that PCT announces) in PCT announcement WO 2005/018579, and it is openly incorporated at this by reference.
The glucose dehydrogenase that non-natural exists can use known method such as, such as, and mutagenesis, orthogenesis and similar approach preparation.Have the GDH enzyme of suitable activity, no matter be naturally occurring or non-natural existence, PCT can be used to announce the mensuration described in the embodiment 4 of WO 2005/018579 and easily differentiate, the open of described patent is incorporated at this by reference.The glucose dehydrogenase that exemplary non-natural exists is announced in WO 2005/018579 at PCT and is provided as SEQ ID NO:62,64,66,68,122,124 and 126.Encode their polynucleotide sequence is announced in WO2005/018579 at PCT and is provided as SEQ ID NO:61,63,65,67,121,123 and 125 respectively.All these sequences are incorporated at this by reference.The glucose dehydrogenase that the other non-natural being suitable for using in the enzymatic reduction reaction of ketone body powder disclosed herein exists provides in U.S. Application Publication the 2005/0095619th and No. 2005/0153417, and it is openly incorporated at this by reference.
The glucose dehydrogenase used in the enzymatic reduction reaction of ketone body powder described herein is announced at PCT in the mensuration described in the embodiment 4 of WO 2005/018579 and can be shown at least about 10 μm of ol/min/mg, and is sometimes at least about 10 2μm ol/min/mg or about 10 3μm ol/min/mg, up to about 10 4the activity of μm ol/min/mg or higher.
The enzymatic reduction reaction of ketone body powder described herein is generally carried out in a solvent.Suitable solvent comprises water, organic solvent (such as ethyl acetate, butylacetate, 1-octanol, heptane, octane, methyl tertiary butyl ether (MTBE), toluene and similar solvent), ionic liquid (such as 1-ethyl 4-methylimidazole a tetrafluoro borate, 1-butyl-3-methyl imidazolium tetrafluoroborate, 1-butyl-3-Methylimidazole hexafluorophosphate and similar ionic liquid).In some embodiments, use aqueous solvent, comprise water and water-based co-solvent system.
Exemplary water-based co-solvent system has water and one or more organic solvents.In general, the organic solvent constituent of water-based co-solvent system is selected can not to make ketoreductase deactivation completely to make it.Utilize as described herein those enzyme assaies, by the enzymatic activity of engineering ketoreductase of specifying with the concern substrate measurement determined in candidate's solvent systems, can easily differentiate suitable co-solvent system.
The organic solvent constituent of water-based co-solvent system can be miscible with aqueous components, provides single liquid phase, can be maybe and aqueous components partial miscibility or not miscible, provide two liquid phases.In general, when making use co-solvent system, select it to be two-phase, water-dispersion in organic solvent, or on the contrary.In general, when utilizing water-based co-solvent system, need to select can easily with the organic solvent of aqueous phase separation.In general, in co-solvent system water than organic solvent ratio usually organic solvent than water about 90: 10 in the scope of about 10: 90 (v/v), and organic solvent than water between 80: 20 and 20: 80 (v/v).Co-solvent system can be pre-formed before adding reaction mixture, or it original position can be formed in reactor.
Aqueous solvent (water or water-based co-solvent system) can be pH buffering or not cushion.In general, about 10 or following pH, usually can reduce in the scope of about 5 to about 10.In some embodiments, about 9 or following pH, usually reduce in the scope of about 5 to about 9.In some embodiments, about 8 or following pH, usually reduce in the scope of about 6 to about 8 in the scope of about 5 to about 8 of being everlasting.Can also about 7.8 or following, or 7.5 or following pH under reduce.Alternatively, can reduce under neutral pH (namely about 7).
During reduction reaction process, the pH of reaction mixture can change.By adding acid or alkali during reaction process, the pH of reaction mixture can be maintained within the scope of required pH or required pH.Alternatively, the aqueous solvent comprising buffer reagent by use can control pH.The appropriate buffer maintaining required pH scope is known in the art, and comprises, such as, and phosphate buffer, trolamine buffer reagent and similar buffer reagent.Also can use the combination of the interpolation of buffering and acid or alkali.
When using glucose/glucose dehydrogenase cofactor regeneration system, if the water-based glyconic acid produced is not neutralized by other approach, representated by reaction formula (1), the common generation of glyconic acid (pKa=3.6) induces reaction the decline of pH of miscellany.By standard buffers technology, wherein in buffer reagent and glyconic acid until the buffer capacity that provides, or by synchronously adding alkali with conversion process, the pH of reaction mixture is maintained desired level.Also can use the combination of the interpolation of buffering and alkali.The appropriate buffer maintaining required pH scope is described above.For in and the suitable alkali of glyconic acid be organic bases as amine, alkoxide and similar organic bases, and inorganic basis is as hydroxide salt (such as NaOH), carbonate (such as NaHCO 3), supercarbonate (such as K 2cO 3), subphosphate (such as K 2hPO 4, Na 3pO 4) and similar mineral alkali.Alkali with conversion process adds, and manually can carry out, or more easily, carry out by using automatic titrimeter as permanent pH device (pH stat) while monitoring reaction mixture pH.The combination of the interpolation of partial buffer capacity and alkali also can be used for process control.
When in using the interpolation of alkali and discharge during the enzymatic reduction reaction of ketone body powder glyconic acid time, the process of conversion is monitored by the amount of the alkali maintaining pH and add.Usually, by reduction process, join non-buffered or the alkali of reaction mixture of partial buffer add with aqueous solution.
In some embodiments, cofactor regeneration system can comprise hydrogenlyase.Herein, term " hydrogenlyase " and " FDH " are used interchangeably to refer to NAD +or NADP +dependent enzyme, they are catalysis formate and NAD respectively +or NADP +be converted into carbonic acid gas and NADH or NADPH.The hydrogenlyase being suitable for the cofactor regeneration system be used as in the enzymatic reduction reaction of ketone body powder described herein comprises the hydrogenlyase of naturally occurring hydrogenlyase and non-natural existence.Hydrogenlyase comprises announces the SEQ ID NO:70 (pseudomonas (Pseudomonas sp.)) of WO 2005/018579 and the hydrogenlyase of 72 (Candida boidinii (Candida boidinii)) corresponding to PCT, SEQ ID NO:70 and 72 is encoded respectively by the polynucleotide sequence of the SEQ ID NO:69 and 71 corresponding to PCT announcement 2005/018579, and the open of described patent is incorporated at this by reference.The hydrogenlyase used in method described herein, no matter be naturally occurring or non-natural existence, can show at least about 1 μm of ol/min/mg, sometimes at least about 10 μm of ol/min/mg or at least about 10 2μm ol/min/mg, up to about 10 3the activity of μm ol/min/mg or higher, and announce at PCT in the mensuration described in the embodiment 4 of WO 2005/018579 and easily can screen that it is active.
As used herein, term " formate " nail acid anion (HCO 2 -), formic acid (HCO 2h) and composition thereof.Formate can be provided as the form of salt, normally alkali metal salts or ammonium salt (such as HCO 2na, KHCO 2nH 4and similar salt); The form of formic acid, normally aqueous formic acid; Or the form of its mixture.Formic acid is moderate acid.In the aqueous solution in several pH units of its pKa (in water pKa=3.7), formate is with the HCO of equilibrium concentration 2 -and HCO 2h exists.When pH value is greater than about pH 4, formate is mainly with HCO 2 -exist.When formate provides as formic acid, reaction mixture is usually buffered or reduces to provide required pH by adding alkali to make acidity, normally about pH 5 or higher.For in and the suitable alkali of formic acid include but not limited to that organic bases is as amine, alkoxide and similar organic bases, and inorganic basis is as hydroxide salt (such as NaOH), carbonate (such as NaHCO 3), supercarbonate (such as K 2cO 3), subphosphate (such as K 2hPO 4, Na 3pO 4) and similar mineral alkali.
For the pH value being greater than about pH 5, now formate is mainly with HCO 2 -exist, following reaction formula (2) describes the formate of formate dehydrogenase enzyme catalysis to NAD +or NADP +reduction.
When using formate and hydrogenlyase as cofactor regeneration system, by standard buffers technology, wherein buffer reagent release proton is at most provided buffer capacity, or by adding acid with conversion process simultaneously, the pH of reaction mixture is maintained required level.The suitable acid adding to maintain pH during reaction process comprises organic acid, such as carboxylic acid, sulfonic acid, phosphonic acids and similar organic acid, mineral acid is haloid acid (all example hydrochloric acids), sulfuric acid, phosphoric acid and similar mineral acid such as, and acid salt is dihydrogen orthophosphate (such as KH such as 2pO 4), hydrosulfate (such as NaHSO 4) and similar acid salt.Some embodiments utilize formic acid, maintain the pH of concentration of formate greater and solution whereby.
During the reduction reaction using formate/hydrogenlyase cofactor regeneration system, when using interpolation acid to maintain pH, by maintaining the amount monitoring conversion process of the acid that pH adds.Usually, the acid of join in conversion process non-buffered or partial buffer reaction mixture is added with aqueous solution.
Herein, term " dehydrogenating para-alcohol enzyme " and " sADH " exchange and use to refer to NAD +or NADP +dependent enzyme, they are catalysis secondary alcohol and NAD respectively +or NADP +be converted into ketone and NADH or NADPH.Below, reaction formula (3) describes secondary alcohol to NAD +or NADP +reduction, for Virahol.
The dehydrogenating para-alcohol enzyme being suitable for the cofactor regeneration system be used as in the enzymatic reduction reaction of ketone body powder described herein comprises the dehydrogenating para-alcohol enzyme of naturally occurring dehydrogenating para-alcohol enzyme and non-natural existence.Naturally occurring dehydrogenating para-alcohol enzyme comprises the known alcoholdehydrogenase from the hot anerobe of Bu Shi (Thermoanerobiumbrockii), rhodococcus erythropolis (Rhodococcus etythropolis), lactobacillus kefir and short lactobacillus, but not naturally occurring dehydrogenating para-alcohol enzyme comprises engineering alcoholdehydrogenase derivative thus.The dehydrogenating para-alcohol enzyme used in method described herein, no matter be naturally occurring or non-natural existence, can show at least about 1 μm of ol/min/mg, sometimes at least about 10 μm of ol/min/mg or at least about 10 2μm ol/min/mg, up to about 10 3the activity of μm ol/min/mg or higher.
Suitable secondary alcohol comprises rudimentary secondary alkanol and aryl-alkyl methyl alcohol.The example of rudimentary secondary alcohol comprises Virahol, 2-butanols, 3-methyl-2-butanols, 2-amylalcohol, 3-amylalcohol, 3,3-dimethyl-2-butanols and similar rudimentary secondary alcohol.In one embodiment, secondary alcohol is Virahol.Suitable aryl-alkyl methyl alcohol comprises 1-aryl alcohol that is unsubstituted and that replace.
When using secondary alcohol and dehydrogenating para-alcohol enzyme as cofactor regeneration system, be ketone by dehydrogenating para-alcohol enzyme by secondary alcohol coupled oxidation, and then the NAD that reduction produces +or NADP +.Some engineering ketoreductases also have the activity making the dehydrogenation of secondary alcohol reductive agent.Use secondary alcohol as in the embodiment of reductive agent at some, engineering ketoreductase and dehydrogenating para-alcohol enzyme are same enzymes.
When using cofactor regeneration system to perform the embodiment of the enzymatic reduction reaction of ketone body powder described herein, just begin to provide the cofactor of oxidation or reduction form.As mentioned above, the cofactor of oxidation is converted into its reduction form by cofactor regeneration system, then in the reduction of ketoreductase substrate, uses described reduction form.
In some embodiments, cofactor regeneration system is not used.The reduction reaction performed for not using cofactor regeneration system, adds the cofactor of reduction form to reaction mixture.
In some embodiments, when using the full cell of host organisms to perform this process, but this full cell self provides cofactor.Alternatively or in combination, but this cell self or restructuring glucose dehydrogenase is provided.
In the reaction of execution Stereoselective reduction described herein, purifying enzyme can be added, with the full cell of gene transformation of this enzyme of encoding and/or the engineering ketoreductase of the cell extract of this cell and/or the form of lysate and any enzyme comprising optional cofactor regeneration system to reaction mixture.The gene of coding engineering ketoreductase and optional cofactor regeneration enzyme can be transformed into host cell respectively, or is transformed into identical host cell together.Such as, in some embodiments, the gene transformation of one group of host cell available code engineering ketoreductase, and the gene transformation of another group available code cofactor regeneration enzyme.With the form of the form of full cell or lysate derivative thus or extract, the cell one that two groups transform can be used from reaction mixture.In other embodiments, host cell can use the gene transformation of coding engineering ketoreductase and cofactor regeneration enzyme.
With the full cell of the gene transformation of encode engineering ketoreductase and/or optional cofactor regeneration enzyme, or its cell extract and/or lysate, can use with multiple different form, comprise solid (such as lyophilized solid, spray-dired solid and similar solid) or semisolid (such as thick paste).
By precipitation (ammonium sulfate, polymine, thermal treatment or similar process), subsequently can partial purification cell extract or cell lysates by the desalination program (such as ultrafiltration, dialysis and similar process) before freeze-drying.Be cross-linked by using known linking agent (such as such as glutaraldehyde) or be fixed on solid phase (such as Eupergit C and similar solid phase) and stablize any cell product.
Various multi-form solid reactant (such as enzyme, salt etc.) can be provided to reaction, comprise powder (such as freeze-drying, spray-dired and similar), solution, emulsion, suspension and similar type.Use the method and apparatus known to those of ordinary skill in the art, can easily freeze-drying or spraying dry reactant.Such as, protein soln can be freezing at-80 DEG C with little equal portions, then joins in the cryodesiccation chamber of precooling, subsequently application of vacuum.After water in removing sample, usually temperature is risen to 4 DEG C, continue 2 hours, then discharge vacuum and reclaim freeze-drying sample.
The amount of reactant used in reduction reaction usually by according to the amount of required product and concomitantly ketoreductase substrate used amount and change.Below instruct the amount that can be used for ketoreductase, cofactor and the optional cofactor regeneration system determining to use.In general, use about 50mg to the cofactor of the ketoreductase of about 5g and about 10mg extremely about 150mg, can the ketone substrate of working concentration about 20 to 300 grams per liter.Will be readily appreciated by those of ordinary skill in the art that and how to change this tittle they to be adjusted to productivity and production-scale desired level.According to the amount of cofactor used and/or ketoreductase, the suitable amount of optional cofactor regeneration system easily can be determined by normal experiment.In general, the horizontal exceeding ketoreductase substrate of the reductive agent (such as glucose, formate, Virahol) of use etc. mol level, transform completely substantially completely or almost to realize ketoreductase substrate.
The addition sequence of reactant is unimportant.Reactant can join in solvent (such as single-phase solvent, two-phase water-based co-solvent system and similar solvent) in the identical time together, or alternatively, can add some in reactant respectively, and some reactants add together at different time points.Such as, can first cofactor regeneration system, cofactor, ketoreductase and ketoreductase substrate be joined in solvent.
In order to improve mixing effect when making use co-solvent system, can first cofactor regeneration system, ketoreductase and cofactor be added and be mixed into aqueous phase.Then can add organic phase and mix, adding ketoreductase substrate subsequently.Alternatively, ketoreductase substrate can before joining aqueous phase, pre-mixing in organic phase.
Conditions suitable for performing the enzymatic reduction reaction of ketone body powder described herein comprises a variety of condition, they can easily be optimized by normal experiment, experiment includes but not limited to make engineering ketoreductase and substrate contact under experiment pH and temperature, and such as, use the method described in embodiment provided herein to detect product.
Usually in the temperature range of about 15 DEG C to about 75 DEG C, the enzymatic reduction of ketone body powder is performed.For some embodiments, in the temperature range of about 20 DEG C to about 55 DEG C, perform reaction.In other embodiments, in the temperature range of about 20 DEG C to about 45 DEG C, reaction is performed.Reaction can also be performed at ambient conditions.
Usual permission reduction reaction is carried out, until realize reducing completely substantially completely or almost of substrate.Substrate is reduced to product can use known method, is monitored by detection substrate and/or product.Suitable method comprises gas-chromatography, HPLC and similar approach.The conversion yields of the alcohol reduzate produced in the reactive mixture is greater than about 50% usually, also can be greater than about 60%, also can be greater than about 70%, also can be greater than about 80%, also can be greater than 90%, and is usually greater than about 97%.
8. embodiment
In following representative embodiment, illustrate various characteristic sum embodiment of the present disclosure, embodiment is intended to example instead of restriction.
8.1 embodiments 1: the acquisition of wild-type ketoreductase gene and the structure of expression vector
According to the aminoacid sequence of the ketoreductase reported with as U.S.Provisional Serial 60/848,950 and the codon optimized algorithm described by embodiment 1 of WO2008042876 (being incorporated at this by reference), be designed for ketoreductase (KRED) encoding gene at expression in escherichia coli.Use the oligonucleotide synthetic gene comprising 42 Nucleotide, and be cloned in the expression vector pCK110900 (described by Fig. 3 of U.S. Patent Application Publication 20060195947) under the control of lac promotor.This expression vector also comprises P15a replication orgin and chloramphenicol resistance gene.Use standard method by the Plastid transformation of generation to intestinal bacteria W3110.The sequence of codon optimized gene and the polypeptide of coding list in table 3.As U.S.Provisional Serial 60/848, described by 950, confirm the activity of wild-type ketoreductase.
Disclosed in code book, the polynucleotide of engineering ketoreductase are similarly cloned in carrier pCK110900, to express in intestinal bacteria W3110.
8.2 embodiments 2: the generation of ketoreductase powder; Shaking flask step.
The LB meat soup (LuriaBertani broth) of 30 μ g/ml paraxin and 1% glucose will be contained in 50ml containing the colibacillary single microorganism colony inoculation of plasmid with paid close attention to ketoreductase gene.30 DEG C, under 250rpm vibration, make cell grow overnight (at least 16 hours) in incubator.Culture is diluted in 250ml Terrific meat soup (12g/L bacto-tryptone, 24g/L yeast extract, 4ml/L glycerine, 65mM potassiumphosphate, pH 7.0,1mM MgSO 4, 30 μ g/ml paraxin are in 1 liter of flask) be 0.2 to the optical density(OD) (OD600) at 600nm place, and allow it to grow at 30 DEG C.When the OD600 of culture is 0.6 to 0.8, the expression of ketoreductase gene is induced with 1mM IPTG, and overnight incubation (at least 16 hours).By centrifugal (5000rpm, 15min, 4 DEG C) harvested cell, and abandoning supernatant.By 100mM trolamine (muriate) damping fluid of cell precipitation thing with isopyknic cold (4 DEG C), pH 7.0 (when ADH-LK and ADH-LB and engineering ketoreductase derivative thus, comprises 2mM MgSO 4) again suspend, and by harvested by centrifugation as above.The cell of washing is again suspended in cold trolamine (muriate) damping fluid of two volumes, and pass French press 2 times under 12000psi, and remain on 4 DEG C.By centrifugal (9000rpm, 45min, 4 DEG C) removing cell debris.Collect the lysate supernatant liquor of clarification, and store at-20 DEG C.The freezing clarified lysate of freeze-drying provides the dried powder of thick ketoreductase.
As U.S.Provisional Serial 60/848, described by 950, confirm the activity of wild-type ketoreductase.To 1mL 100mM phosphoric acid salt (sodium) damping fluid, the solution of pH 7.5 adds 10mg ketoreductase powder, 50mg NAD (P) H, 100 μ L Virahols and 10mg 4 '-chloro-acetophenone or unsubstituted phenyl methyl ketone.Reaction mixture is at room temperature stirred 16 hours, then with 1mL MTBE extraction.By the conversion of 4 '-chloro-phenyl methyl ketone in the sample of chiral HPLC MTBE phase and the enantiomer composition of product.
8.3 embodiments 3: the production-fermentation step of ketoreductase.
In the 15L fermentor tank of air agitation, make the growth medium of 6.0L reach the temperature of 30 DEG C, described substratum comprises the Trisodium Citrate of 0.88g/L ammonium sulfate, 0.98g/L; 12.5g/L the trace element solution of dipotassium hydrogen phosphate trihydrate, the potassium primary phosphate of 6.25g/L, the Tastone-154 yeast extract of 6.2g/L, 0.083g/L ferric ammonium citrate and 8.3ml/L, trace element solution contains the calcium chloride dihydrate of 2g/L, 2.2g/L ZINC SULFATE HEPTAHYDRATE, 0.5g/L magnesium sulfate monohydrate, 1g/L cuprous sulfate heptahydrate, 0.1g/L ammonium molybdate tetrahydrate and 0.02g/L sodium tetraborate decahydrate.The fermentor tank rear phase index culture inoculation of intestinal bacteria W3110, described intestinal bacteria W3110 contains the plasmid with paid close attention to ketoreductase gene, and in shaking flask as described in Example 2, grow to initial OD600 is 0.5 to 2.0.Fermentor tank stirs with 500-1500rpm, and provides air to keep 30% saturated dissolved oxygen level or higher with 1.0-15.0L/min to fermenting container.By adding the ammonium hydroxide of 20%v/v, the pH of culture is controlled 7.0.The growth of maintain thing is carried out by the feed liquid of adding containing 500g/L cerelose (cerelose), 12g/L ammonium chloride and 10.4g/L magnesium sulfate 7 hydrate.After culture reaches the OD600 of 50, induce the expression of ketoreductase by adding isopropyl-β-D-thiogalactoside(IPTG) (IPTG) to the ultimate density of 1mM.Culture is made to grow other 14 hours.Then culture is cooled to 4 DEG C, and maintains 4 DEG C until results.At 4 DEG C in Sorval RC12BP whizzer with 5000G centrifugal 40 minutes harvested cells.The cell of results is directly used in following downstream recovery process, or stores at 4 DEG C until this type of uses.
At 4 DEG C, cell precipitation thing is suspended in again 100mM trolamine (muriate) damping fluid of 2 volumes, pH 6.8 is to the wet cell mashed prod of every volume.By using the pressure of 12000psig by the refiner of this suspension by installation secondary homogenate valve module, ketoreductase in cell is discharged from cell.After destruction, immediately cell homogenates thing is cooled to 4 DEG C.By the polyethylenimine solution of 10%w/v, pH 7.2 joins lysate to ultimate density 0.5%w/v, and stirs 30 minutes.By in standard laboratory whizzer with 5000G centrifugal 30 minutes, the suspension of generation is clarified.Incline and the supernatant liquor of clarification, use and there is concentrated 10 times of the cellulosic ultrafiltration membrane that molecular weight cut-off is 30Kd.Final enriched material is scattered in kiver, freezing at-20 DEG C, and be lyophilized into powder.By ketoreductase powder storage in-80 DEG C.
8.4 embodiments 4: the analytical procedure determining the conversion of 3-ketone group tetramethylene sulfide and the enantiomeric excess of R-3-hydroxy tetrahydro thiophene.
Determine the chirality GC transformed: use Agilent HP-5MS gas-chromatography to determine that 3-ketone group tetramethylene sulfide is reduced into 3-hydroxy tetrahydro thiophene, described gas-chromatography is equipped with 5% phenylmethylsiloxane post (model 19091S-433:30mx250 μm, nominal thickness 0.25 μm), there is the helium flow amount of 1.0mL/min.Temperature in is 220 DEG C, and uses following temperature program(me): 120 DEG C, continues 1 minute, then 20 DEG C/min to 160 DEG C, and then 160 DEG C continue 0.5 minute.210nm place, spectrophotometric detection by quantitative compound.The retention time of ketone and alcohol is 2.8 minutes and 3.0 minutes respectively.
Determine the chirality HPLC of the stereoisomeric purity of 3-hydroxy tetrahydro thiophene: at room temperature use Agilent 1100 or 1050HPLC to determine the abundance of R and the S enantiomorph of 3-hydroxy tetrahydro thiophene; described HPLC is equipped with Chiralpak AD post (4.6x250mm; and unprotect post); there is IPA/ hexane (2/98) as elutriant, flow 2.5mL/min.The retention time of ketone, S-alcohol and R-alcohol is 8.6,12.0 and 13.1 minutes respectively.
Alternatively, can use the HPLC that Chiralpak AD-H post (4.6x150mm and 10mm guard column) is housed at 40 DEG C, it uses IPA/ hexane (2/98), flow 2.0mL/min.210nm place, spectrophotometric detection by quantitative compound.The retention time of ketone, S-alcohol and R-alcohol is 6.3,8.7 and 9.4 minutes respectively.
8.5 embodiments 5: wild-type ketoreductase is for reducing the assessment of 3-ketone group tetramethylene sulfide
Under following cofactor restricted condition, screening is used for the ketoreductase (embodiment 1) described in the table 3 of enantioselective reduction 3-ketone group tetramethylene sulfide: under air, and 1-20mg enzyme, 1mL cofactor stock solution containing 766mg NAD (P) H be dissolved in 20mL100mM pH 8.0 trolamine (muriate) damping fluid and 20 μ L 3-ketone group tetramethylene sulfides are added bottle.At room temperature stir after spending the night, with ethyl acetate abstraction reaction mixture, and measure enantioselectivity according to embodiment 4 is described.The results are described in table 4.(NR=does not react.)
This embodiment is presented in the wild-type enzyme of test, and ADH-LK is the ketoreductase of most R-enantioselectivity, and it produces the R-3-hydroxy tetrahydro thiophene of 67%e.e..Therefore, ADH-LK is chosen as the starting point of the enzyme engineering for required feature.
8.6 embodiments 6: the high-throughput NADPH fluorescence prescreen differentiating the enzyme reduction 3-ketone group tetramethylene sulfide improved.
Be transformed to intestinal bacteria W3110 containing the plasmid library of ketoreductase gene of evolving and coated plate on Luria-Bertani (LB) agar plate containing 1% glucose and 30 μ g/mL paraxin (CAM).Hatch at least 16 hours at 30 DEG C after, use Q- automatic bacterial colony picking device (Genetix USA, Inc., Beaverton, OR), by bacterium colony picking to the 96 hole shallow bore hole microtiter plates containing 180 μ L LB, 1% glucose and 30 μ g/mL CAM.Under 250rpm vibration, make cell grow overnight at 37 DEG C.Then, this culture of 10 μ L is transferred on the 96 hole microtiter plates (deep hole) containing 390 μ LTerrific meat soups (TB) and 30 μ g/mL CAM.30 DEG C, under 250rpm vibration, deep-well plates is hatched 2.5 to 3 hours (OD 6000.6-0.8), after, recombination is expressed by isopropylthiogalactoside (IPTG) the inducing cell culture of ultimate density 1mM.Then, 30 DEG C, under 250rpm vibration, by plate overnight incubation.
Cell, via centrifugation, dissolves in damping fluid at 300 μ L and again suspends, and within least 2 hours, dissolve by room temperature vibrating.Dissolve damping fluid and contain 100mM trolamine (muriate) damping fluid, pH 7.0-7.2,1mg/mL N,O-Diacetylmuramidase and 750 μ g/mL Polymyxin B-sulfate USPs.Plate centrifugal 10 minutes at 4000RPM, and in fluorometric assay, use the supernatant liquor (lysate) of clarification.
In 96 hole lucifuge microtiter plates, 20 μ l clarified supernatant (if desired, are diluted in 100mM trolamine/muriate damping fluid, pH 7.0,1mM MgSO 4) join 180 μ l measure mixtures, described mensuration mixture by 100mM trolamine (muriate) damping fluid, pH 7.0,1mM MgSO 4, 0.25mM NADPH, 600mM glucose, 1200mM gluconic acid sodium salt and 1mM 3-ketone group tetramethylene sulfide composition, and by basis at Flexstation (Molecular Devices, USA) in, after exciting under 330nm, the reduction of the fluorescence of NADPH under 445nm, measures reaction process.In order to assess the thermostability of KRED variant, clarified supernatant is under the temperature range of 40 DEG C to 50 DEG C, and optionally preincubate at least 16 hours, then joins and measure in mixture.
This example describes the method for differentiating the KRED variant that 3-ketone group tetramethylene sulfide rate of reduction improves and/or thermostability improves.
8.7 embodiments 7: use the ketoreductase for the reduction of 3-ketone group tetramethylene sulfide of the glucose/glucose dehydrogenase of cofactor recirculation high-throughput HPLC that is active and enantioselectivity to measure
Lysate is prepared described by embodiment 6.By the cell lysates of measured quantity being transferred to each hole of deep hole microtiter plate, measure ketoreductase active.Mensuration mixture (final volume in each hole is 500 μ l) in hole is containing 1mg/mL GDH, 0.4mg/ml Na-NADP, 0.2 to 1.25M glucose, 0 to 400mM gluconic acid sodium salt, 100mM trolamine (muriate) damping fluid (pH 7), 20mM MgSO 4with nearly 50mg calcium carbonate.Reaction is started by adding nearly 100 μ l cell lysates and 50 μ l 3-ketone group tetramethylene sulfide (ultimate density 1M).After aluminium/polypropylene sheet temperature resistant encapsulation band (Velocity 11 (Menlo Park, CA), catalog number 06643-001) sealing, plate is hatched at 4 DEG C to 25 DEG C nearly 16 hours.1ml ethyl acetate is expelled to each hole of plate, and plate is shaken 10 minutes, then centrifugal 20 seconds; By 100 μ l ethyl acetate phase transitions to down-the-hole microtiter plate.The sealing of these sample panel temperature resistant encapsulation bands is to avoid evaporating.Sample is analyzed by HPLC by the method for embodiment 4.
How the display of this embodiment is differentiated the activity with the improvement of reduction 3-ketone group tetramethylene sulfide in ketoreductase gene library and/or is formed the ketoreductase variant of enantioselectivity of improvement of R-3-hydroxy tetrahydro thiophene.
8.8 embodiments 8: derived from the engineering ketoreductase of ADH-LK to the reduction of 3-ketone group tetramethylene sulfide.
Derived from the ketoreductase of the improvement of ADH-LK variant with following preparative-scale evaluation.18ml water (water or damping fluid) containing 6.63g glucose is added in 25ml tri-neck container, this container is equipped with the magnetic stirring bar of PTFE parcel and is connected to the pH electrode of automatic titrator, and it controls to add for the pH carrying out alkali through conduit to container as required.Add 3.0g 3-ketone group tetramethylene sulfide (vapor enrichment), 30 to the 120mg KRED variant in the aqueous phase of 1mL, the 30mg GDH in the aqueous phase of 1mL and 12mg NADP-Na subsequently 2.PH is maintained 7 by adding 4NNaOH by automatic titrator, and it is continued record.By the speed of alkali with constantly add, and to the periodic sampling of reaction mixture to extract by ethyl acetate, and by the methods analyst of embodiment 4, monitoring reaction process.When having reacted, the concentration of R-3-hydroxy tetrahydro thiophene is 100 ± 5g/L.
Table 5 provide corresponding ketoreductase variant SEQ ID NO., from the quantity of the amino acid mutation of wild-type ADH-LK, they are for reducing the activity (as 3-ketone group tetramethylene sulfide changed into completely the amount of the enzyme powder needed for 3-hydroxy tetrahydro thiophene reaction in 24 hours) of 3-ketone group tetramethylene sulfide and the R-enantioselectivity (as e.e. scope) of 3-hydroxy tetrahydro thiophene product.
This embodiment illustrates the enantioselectivity providing the improvement contrasted with wild-type ketoreductase ADH-LK derived from ketoreductase ADH-LK and active engineering ketoreductase.
aenantioselectivity arranges:
0 (R) enantiomorph 61.0-79.99%ee
+ (R) enantiomorph 80.0-89.99%ee
++ (R) enantiomorph 90.0-94.99%ee
+++ (R) enantiomorph 95.0-97.99%ee
++++(R) enantiomorph 98.0-98.99%ee
+++ ++ (R) enantiomorph > 99.0%ee
bactive row:
0 needs this enzyme of the concentration of 1.0-4.0g/L to be provided in 20 to 24h with this enzyme being provided in 24h the concentration transforming 100g/L 3-ketone group tetramethylene sulfide+0.8-1.0g/L completely transforms 100g/L 3-ketone group tetramethylene sulfide completely ++ and this enzyme of the concentration of 0.8-1.0g/L is provided in 12 to 20h and transforms 100g/L 3-ketone group tetramethylene sulfide completely
cstability arranges:
-than the poor stability of ADH-LK
0 is similar to the stability of ADH-LK
+ more stable than ADH-LK
The preparative-scale of 8.9 embodiment 9:R-(3)-hydroxy tetrahydro thiophene is produced
Under air and room temperature, by 600mL 100mM pH 7.0 trolamine (muriate) damping fluid, 2mL 1M MgSO 4join in the three-necked flask of 2-L jacketed with 225g D-Glucose, this flask is equipped with the mechanical stirrer of about 300rpm, thermometer and is connected to the pH electrode of automatic titrator, and it controls to add for the pH carrying out alkali through conduit to container as required.After glucose dissolves (about 1/2h), 100g 3-ketone group tetramethylene sulfide is added reaction to obtain two-phase mixture.Temperature of reaction is reduced to 15 DEG C, and be added in the 1.0g ketoreductase powder (SEQ-ID No.22) in 30mL 100mM pH 7.0 trolamine (muriate) damping fluid and the 0.25g GDH in 20mL 100mM pH7.0 trolamine (muriate) damping fluid, and the 0.4g NADP-Na in 10mL 100mM pH7.0 trolamine (muriate) damping fluid 2.Temperature of reaction is through outer circulation condenser at 15 DEG C, and pH is maintained 7.0+0.1 by adding 8N NaOH by automatic titrator.Stir 15h at 15 DEG C after, circulator temperature is risen to 25 DEG C, and after other 1 hour, add 0.25g GDH powder (final GDH concentration=0.5g/L).Altogether after 23 hours, add 10g Na 2s 2o 5.Stir in addition after 1 hour at 25 DEG C, extract product by 750ml ethyl acetate.By the organic phase of about 20g diatomite 545 filtering separation.To wet Celite pad with 750mL EtOAc rinsing.The organic phase 100mL water washing merged.At 60 DEG C, by the organic phase rotary evaporation in vacuo be separated, to obtain the 3-hydroxy tetrahydro thiophene of faint yellow oily 83.6g (yield 82%).Chiral HPLC shows (R)-3-hydroxy tetrahydro thiophene of 99.3% enantiomeric excess.
For all objects, all publications that the application quotes, patent, patent application and alternative document are incorporated to its entirety by reference at this, it is equal to for all objects, indicates each individual publication, patent, patent application or alternative document individually and is incorporated to by reference.
Although set forth and described each specific embodiment, should be understood that and can carry out various change and not depart from the spirit and scope of the present invention.
Sequence table
<110> Jack beam
History carries Pfennig J Jane
EMILY Mu Defu
La Mawolan Delhi
James La Lunde
Ji Jiatehasiman
 
The Ketoreductase polypeptides of improvement prepared by the stereoselectivity that <120> is used for (R)-3-hydroxy tetrahydro thiophene
<130>376247-016(385337)
<150>US 60/957,974
<151>2007-08-24
<160>155
 
<210>1
<211>762
<212>DNA
<213> artificial sequence
 
<220>
The short lactobacillus sequence that <223> is codon optimized
 
<400>1
atgtctaacc gtctggatgg caaagtagcc atcattaccg gcgggactct gggtatcggt 60
ttggcaatcg ccacgaaatt tgtagaggag ggtgcgaaag taatgattac tggtcgtcac 120
tccgatgtag gtgaaaaggc cgccaaatca gtaggcactc cggatcagat tcagtttttt 180
cagcacgatt catccgatga agatggctgg acgaaactgt tcgacgccac cgagaaagca 240
ttcggcccgg ttagcacctt agtgaacaat gcagggattg cagttaacaa aagcgttgaa 300
gaaactacca cggccgaatg gcgtaaactg ctggccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttgaggggtt cgtaggcgat ccgagcctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctgccg 600
ggtgctgagg aagcgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catatatctg tgtgtacctg gcatctaatg aatcgaaatt tgcgacgggt 720
tccgaatttg tggtcgacgg cgggtatacc gcacagtaat ga 762
<210>2
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The translation of the short lactobacillus sequence that <223> is codon optimized
 
<400>2
Met Ser Asn Arg Leu Asp Gly Lys Val Ala Ile Ile Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Thr Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Met Ile Thr Gly Arg His Ser Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Val Gly Thr Pro Asp Gln Ile Gln Phe Phe Gln His Asp Ser
50 55 60
Ser Asp Glu Asp Gly Trp Thr Lys Leu Phe Asp Ala Thr Glu Lys Ala
65 70 75 80
Phe Gly Pro Val Ser Thr Leu Val Asn Asn Ala Gly Ile Ala Val Asn
85 90 95
Lys Ser Val Glu Glu Thr Thr Thr Ala Glu Trp Arg Lys Leu Leu Ala
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Phe Val Gly Asp Pro Ser Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Pro Gly Ala Glu Glu Ala Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Tyr Ile Cys Val Tyr Leu Ala Ser Asn Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ser Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210>3
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The lactobacillus kefir sequence that <223> is codon optimized
 
<400>3
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttgaggggtt cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>4
<211>252
<212>PRT
<213> artificial sequence
                  
<220>
The translation of the lactobacillus kefir sequence that <223> is codon optimized
 
<400>4
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>5
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>5
atgaccgatc gtctgaaaag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acatcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>6
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>6
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser His
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>7
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>7
atgaccgatc gtctgaagaa caaagtagcc atcgtaaccg gcgggactct gggtataggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgtttccaa aagcgttgaa 300
gacactacca cggaggattg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>8
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
<400>8
Met Thr Asp Arg Leu Lys Asn Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Asp Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>9
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>9
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggaccct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgag ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgcaatc acggcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>10
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>10
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Arg Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Gln Ser Arg
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>11
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>11
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gcgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
<210>12
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>12
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>13
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>13
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>14
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>14
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>15
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>15
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctagtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>16
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>16
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>17
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>17
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgacg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgttccaaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggct cgttggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc tgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>18
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>18
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Pro
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Leu Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>19
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>19
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgata catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgttccaaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>20
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>20
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Thr
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Pro
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>21
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>21
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgttccaaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagacca acctggtcga tgatctggaa 600
ggtgctgagg aaatgcaatc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>22
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>22
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Pro
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Asn Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Gln Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>23
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>23
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgttccaaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>24
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>24
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Pro
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>25
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>25
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgttccaaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgcaatc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>26
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>26
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Pro
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Gln Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>27
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>27
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgttccaaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccg acctggtcga tgatctggaa 600
ggtgctgagg aaatgcaatc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>28
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>28
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Pro
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Asp Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Gln Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>29
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
  
<400>29
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgttccaaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccg ggctggtcga tgatctggaa 600
ggtgctgagg aaatgcaatc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>30
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>30
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Pro
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Gly Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Gln Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>31
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>31
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgttgagaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>32
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>32
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Glu
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>33
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>33
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgttgccaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>34
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>34
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Ala
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>35
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>35
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>36
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>36
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>37
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>37
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgcaatc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>38
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>38
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Gln Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>39
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>39
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgtttccag aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgcaatc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>40
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>40
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Ser
85 90 95
Arg Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Gln Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>41
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>41
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgtttccct tagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>42
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>42
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Ser
85 90 95
Leu Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>43
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>43
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>44
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>44
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>45
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>45
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcgaaaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>46
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>46
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Glu
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val AspGly Gly Tyr Thr Ala Gln
245 250
 
<210>47
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>47
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcgagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccactt tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>48
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>48
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Glu
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Phe Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>49
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>49
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cggttcctaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgcaatc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>50
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>50
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Pro
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Gln Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>51
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>51
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cggtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgcaatc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>52
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>52
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Gln Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>53
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>53
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg cgtccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgttccaaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca acgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccga tgggtcactt tggcgagcca 660
aatgatgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>54
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>54
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Pro
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Phe Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>55
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>55
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc tgccaaatca atcggcggta ctgatgttgt ccgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccga aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>56
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>56
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Val Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Glu Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>57
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>57
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgagaaggc cgcccgttca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgttccaaa aagcgttgaa 300
gacactacca cggaggaatg gcgcaaactg atgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctgggtt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgcaatc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg agtcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>58
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>58
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Arg Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Pro
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Met Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Gly Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Gln Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>59
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>59
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgagaaggc cgcccgttca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgcaaactg atgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctgggtt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg agtcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>60
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>60
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Arg Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Met Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Gly Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>61
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>61
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgagaaggc cgcccgttca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgcaaactg atgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctgggtt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgcaatc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg agtcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>62
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>62
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Arg Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Met Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Gly Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Gln Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>63
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>63
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgagaaggc cgcccgttca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagttccaaa aagcgttgaa 300
gacactacca cggaggaatg gcgcaaactg atgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctgggtt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg agtcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>64
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>64
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Arg Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Pro
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Met Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Gly Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>65
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>65
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgagaaggc cgcccgttca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagttccaaa aagcgttgaa 300
gacactacca cggaggaatg gcgcaaactg atgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctgggtt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgcaatc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg agtcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>66
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>66
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Arg Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Pro
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Met Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Gly Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Gln Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>67
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>67
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgagaaggc cgcccgttca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgcaaactg atgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctgggtt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg agtcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>68
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>68
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Arg Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Met Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Gly Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>69
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>69
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgagaaggc cgcccgttca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgcaaactg atgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctgggtt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgcaatc acagcgtacg aaaaccccta tgggceacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg agtcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>70
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>70
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Arg Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Met Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Gly Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Gln Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>71
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>71
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggggact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgttccaaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgcaatc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>72
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>72
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Gly
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Pro
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Gln Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>73
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>73
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggggact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>74
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>74
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Gly
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
<210>75
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>75
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggggact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cggttcctaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgcaatc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>76
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>76
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Gly
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Pro
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Gln Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>77
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>77
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcgggggact gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg tggttcctaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgcaatc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>78
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>78
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Gly
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Val Val Pro
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Gln Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>79
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>79
atgaccgatc gtctgaagag caaagtagcc atcgtaaccg gcggggtcct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgttccaaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgcaatc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>80
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>80
Met Thr Asp Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Val
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Pro
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Gln Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>81
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>81
atgaccgatc gtctgaagca caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>82
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>82
Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>83
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>83
atgaccgatc gtctgaaggg caaagtagcc accgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcatgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgactgt cgtgaacaat gcagggatta cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgcaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttgaggggtt cgtaggcgat ccgacgctgg gggcatacag cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggtgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>84
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>84
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Thr Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Ser Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>85
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>85
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagcgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atccgcaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>86
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>86
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Arg
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>87
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>87
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaac 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>88
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>88
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>89
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>89
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>90
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>90
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>91
<21l>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>91
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atccgcaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>92
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>92
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Arg
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>93
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>93
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgatgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>94
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>94
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Met Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>95
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>95
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta cgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>96
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>96
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Thr Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>97
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>97
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>98
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>98
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>99
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>99
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccactt tggcgaaccg 660
aatgacatcg cgtggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>100
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>100
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Phe Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>101
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>101
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>102
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>102
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>103
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>103
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgagactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>104
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>104
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Arg Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>105
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>105
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccagatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt ttgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>106
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>106
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Arg Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>107
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>107
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgcgaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaacccctg tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>108
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>108
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Val Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>109
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>109
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtggcaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgttgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat cggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcggaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>110
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>110
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Gly Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>111
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>111
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtggcaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cggtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaactg ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atccgtaccc cgctggtcga tggtctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagagtttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>112
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>112
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Gly Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Arg
180 185 190
Thr Pro Leu Val Asp Gly Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>113
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>113
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttgaggggtt cgtaggcgat ccgacgctgg gggcatacag cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgcgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>114
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>114
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Ser Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Ala Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>115
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>115
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttgatgggtt cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>116
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>116
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Asp Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>117
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>117
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catcggatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcgttca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttgaggggtt cgtaggcgat ccgacgctgg gggcatacag cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgcgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggtgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>118
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>118
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Val Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Ser Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>119
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>119
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcatgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgactgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttgaggggtt cgtaggcgat ccgacgctgg gggcatacag cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggtgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>120
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>120
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Ser Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>121
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>121
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcgtca ctgatgttat tcgctttgtc 180
cagcacgacg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttgaggggtt cgtaggcgat ccgacgctgg gggcatacag cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>122
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>122
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Val Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Ser Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>123
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>123
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gtggatgtag gtgaaaaggc cgccagatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattcg gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttgaggggct cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gcatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctgaaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccactt tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>124
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>124
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Val Asp Val Gly Glu Lys Ala Ala
35 40 45
Arg Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Arg Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Leu Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Lys Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Phe Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>125
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>125
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcgggactct gggtgttggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>126
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>126
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Val Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>127
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>127
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcggggctct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttgt tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>128
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>128
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Ala
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Val Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>129
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>129
atgaccgatc gtctgaaggg caaagtagcc atcgtaaccg gcggggctct gggtatcggt 60
ttggcaatcg ccgataagtt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggattg cagtctccaa aagcgttgaa 300
gacactacca cggaggaatg gcgtaaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgac ccgacgttag gggcatacaa cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>130
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>130
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Ala
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>131
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>131
atgaccgatc gtctgaaggg cagagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcattcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgtcac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggca ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta cagcttccaa aagcgttgaa 300
gacactacca cggaggaatg gcataaactg ctgtccgtta atctggatgg tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaaa aataaaggct tgggcgctag catcatcaat 420
atgagcagta ttgaggggtt cgtaggcgat ccgacgctgg gggcatacag cgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggtgaaccg 660
aatgacatcg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>132
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>132
Met Thr Asp Arg Leu Lys Gly Arg Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Phe Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Ala Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Ser Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>133
<211>759
<212>DNA
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>133
atgacctatc gtctgaagag caaagtagcc atcgtaaccg gcgggactct gggtatcggt 60
ttggcaatcg ccgataaatt tgtagaggag ggtgcgaaag tagttattac tggtcgccac 120
gcggatgtag gtgaaaaggc cgccaaatca atcggcggta ctgatgttat tcgctttgtc 180
cagcacgatg catccgatga agcaggctgg acgaaactgt tcgacaccac cgaggaggca 240
ttcggcccgg ttacgaccgt cgtgaacaat gcagggatta ccgtttccaa aagcgttgaa 300
gacactacca cggaggaatg gcacaaacta ctgtccgtta atctggatag tgtttttttc 360
ggcacccgtc tgggcattca gcgcatgaag aataaaggct tgggcgctag catcatcaat 420
atgagcagta tcagtgggtt cgtaggcgat ccgacgctgg gggcatacac tgcttccaag 480
ggggcggtac gtatcatgtc gaaaagcgca gcgctggatt gcgcactgaa ggactacgat 540
gtgcgtgtca acacagtaca tccgggctat atcaagaccc cgctggtcga tgatctggaa 600
ggtgctgagg aaatgatgtc acagcgtacg aaaaccccta tgggccacat tggcgaaccg 660
aatgacgtgg catggatctg tgtgtacctg gcatctgacg aatcgaaatt tgcgacgggt 720
gcagaatttg tggtcgacgg cgggtatacc gcacagtga 759
 
<210>134
<211>252
<212>PRT
<213> artificial sequence
 
<220>
The variant of <223> lactobacillus kefir
 
<400>134
Met Thr Tyr Arg Leu Lys Ser Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Thr Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp His Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Ser Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Ser Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Thr Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Val Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>135
<211>1041
<212>DNA
<213> artificial sequence
 
<220>
<223> is from the codon optimized ADH-SB of reddish brown shadow yeast
 
<400>135
atgaaggcac tgcagtaccg caccatcggc gccccgcccg aggtcgtcac cgtcccggac 60
ccggagccgg gccccggcca ggtgctgttg aaggtgaccg cggccggagt ctgccactcc 120
gacatcgcgg tgatgagctg gcccgccgag ggcttcccgt acgagctgcc gctcaccctc 180
ggccacgagg gcgtcggcac cgtggccgcg ctcggcgccg gggtgacggg gctcgccgag 240
ggcgacgcgg tcgccgtgta cgggccctgg ggctgcggca cctgcgccaa gtgcgcggag 300
ggcaaggaga actactgcct gcgcgccgac gagctgggca tccgtccgcc ggggctcggg 360
cgtccggggt ccatggccga gtacctgctg atcgacgacc cccggcacct ggtcccgctg 420
gacgggctcg acccggtcgc ggcggtgccg ctcaccgacg ccggactgac gccgtaccac 480
gcgatcaagc ggtcgctgcc caagctggtc cccggctcca ccgcggtggt catcggcacc 540
ggtggtctcg gccacgtcgc catccagctg ctgcgcgccc tgacgtccgc ccgggtggtc 600
gccctggacg tcagcgagga gaagctgcgc ctcgcccgtg cggtgggcgc gcacgaggcg 660
gtgctgtcgg acgcgaaggc cgcggacgcg gtgcgcgaga tcaccggcgg tctcggtgcc 720
gaggccgtgt tcgacttcgt cggcgtggcg cccaccgtgc agaccgccgg agccgtcgcg 780
gccgtcgagg gcgatgtcac cctggtcggc atcggcggcg gatcgctgcc cgtcggcttc 840
ggcatgctgc cgttcgaggt gtcggtcaac gccccctact ggggcagccg cagcgagctg 900
accgaggtgc tgaacctggc ccgctccggt gccgtgtcgg tgcacaccga gacgtactcc 960
ctggacgacg ccccgctcgc ctacgagcgg ctgcacgagg gcagggtcaa cggccgcgcg 1020
gtgatcctgc cccacggctg a 1041
 
<210>136
<211>346
<212>PRT
<213> artificial sequence
 
<220>
<223> is from the ADH-SB of reddish brown shadow yeast
 
<400>136
Met Lys Ala Leu Gln Tyr Arg Thr Ile Gly Ala Pro Pro Glu Val Val
1 5 10 15
Thr Val Pro Asp Pro Glu Pro Gly Pro Gly Gln Val Leu Leu Lys Val
20 25 30
Thr Ala Ala Gly Val Cys His Ser Asp Ile Ala Val Met Ser Trp Pro
35 40 45
Ala Glu Gly Phe Pro Tyr Glu Leu Pro Leu Thr Leu Gly His Glu Gly
50 55 60
Val Gly Thr Val Ala Ala Leu Gly Ala Gly Val Thr Gly Leu Ala Glu
65 70 75 80
Gly Asp Ala Val Ala Val Tyr Gly Pro Trp Gly Cys Gly Thr Cys Ala
85 90 95
Lys Cys Ala Glu Gly Lys Glu Asn Tyr Cys Leu Arg Ala Asp Glu Leu
100 105 110
Gly Ile Arg Pro Pro Gly Leu Gly Arg Pro Gly Ser Met Ala Glu Tyr
115 120 125
Leu Leu Ile Asp Asp Pro Arg His Leu Val Pro Leu Asp Gly Leu Asp
130 135 140
Pro Val Ala Ala Val Pro Leu Thr Asp Ala Gly Leu Thr Pro Tyr His
145 150 155 160
Ala Ile Lys Arg Ser Leu Pro Lys Leu Val Pro Gly Ser Thr Ala Val
165 170 175
Val Ile Gly Thr Gly Gly Leu Gly His Val Ala Ile Gln Leu Leu Arg
180 185 190
Ala Leu Thr Ser Ala Arg Val Val Ala Leu Asp Val Ser Glu Glu Lys
195 200 205
Leu Arg Leu Ala Arg Ala Val Gly Ala His Glu Ala Val Leu Ser Asp
210 215 220
Ala Lys Ala Ala Asp Ala Val Arg Glu Ile Thr Gly Gly Leu Gly Ala
225 230 235 240
Glu Ala Val Phe Asp Phe Val Gly Val Ala Pro Thr Val Gln Thr Ala
245 250 255
Gly Ala Val Ala Ala Val Glu Gly Asp Val Thr Leu Val Gly Ile Gly
260 265 270
Gly Gly Ser Leu Pro Val Gly Phe Gly Met Leu Pro Phe Glu Val Ser
275 280 285
Val Asn Ala Pro Tyr Trp Gly Ser Arg Ser Glu Leu Thr Glu Val Leu
290 295 300
Asn Leu Ala Arg Ser Gly Ala Val Ser Val His Thr Glu Thr Tyr Ser
305 310 315 320
Leu Asp Asp Ala Pro Leu Ala Tyr Glu Arg Leu His Glu Gly Arg Val
325 330 335
Asn Gly Arg Ala Val Ile Leu Pro His Gly
340 345
 
<210>137
<211>1032
<212>DNA
<213> artificial sequence
 
<220>
<223> is from the codon optimized ADH-SC of streptomyces coelicolor
 
<400>137
atggcaaaga tcgacaacgc agttctgccg gagggttctc tggtgctggt caccggcgcg 60
aacggctttg tcgctagcca tgtggtcgaa caactgctgg aacacggcta taaggtgcgc 120
ggcactgctc gctctgcctc caaactggcg aacctgcaga aacgttggga cgccaaatac 180
cctggtcgtt tcgagactgc cgttgttgaa gacatgctga agcagggtgc atatgatgaa 240
gttattaaag gcgcggcagg tgtcgcccac atcgcgtccg tggtcagctt ttctaacaaa 300
tatgatgagg tggtaactcc tgcgatcggt ggcacgctga atgccctgcg tgccgcagct 360
gctacgcctt ccgtgaaacg ttttgtgctg accagcagca ctgtttctgc actgattcca 420
aaacctaacg tcgaaggtat ttatctggat gagaagagct ggaacctgga aagcattgat 480
aaggctaaaa ccctgcctga atctgatccg cagaaaagcc tgtgggtcta cgccgcaagc 540
aaaacggaag cggaactggc tgcctggaaa ttcatggacg aaaacaaacc gcactttact 600
ctgaatgccg ttctgccaaa ctacactatc ggtaccattt ttgacccaga aacccaatcc 660
ggttccactt ccggctggat gatgtctctg ttcaatggcg aagtatctcc ggcactggcg 720
ctgatgccgc cgcagtacta tgtctctgca gttgatatcg gtctgctgca cctgggttgt 780
ctggttctgc cgcaaatcga acgccgtcgt gtttacggca ccgcaggcac ctttgattgg 840
aacaccgttc tggcgacctt ccgtaaactg tatccgtcca agacgttccc ggctgacttt 900
ccggatcagg gccaggatct gtccaaattt gataccgccc cgagcctgga gattctgaaa 960
tccctgggcc gccctggctg gcgtagcatc gaggaatcta tcaaagatct ggtgggttcc 1020
gagaccgcct aa 1032
 
<210>138
<211>343
<212>PRT
<213> artificial sequence
 
<220>
<223> is from the ADH-SC of streptomyces coelicolor
 
<400>138
Met Ala Lys Ile Asp Asn Ala Val Leu Pro Glu Gly Ser Leu Val Leu
1 5 10 15
Val Thr Gly Ala Asn Gly Phe Val Ala Ser His Val Val Glu Gln Leu
20 25 30
Leu Glu His Gly Tyr Lys Val Arg Gly Thr Ala Arg Ser Ala Ser Lys
35 40 45
Leu Ala Asn Leu Gln Lys Arg Trp Asp Ala Lys Tyr Pro Gly Arg Phe
50 55 60
Glu Thr Ala Val Val Glu Asp Met Leu Lys Gln Gly Ala Tyr Asp Glu
65 70 75 80
Val Ile Lys Gly Ala Ala Gly Val Ala His Ile Ala Ser Val Val Ser
85 90 95
Phe Ser Asn Lys Tyr Asp Glu Val Val Thr Pro Ala Ile Gly Gly Thr
100 105 110
Leu Asn Ala Leu Arg Ala Ala Ala Ala Thr Pro Ser Val Lys Arg Phe
115 120 125
Val Leu Thr Ser Ser Thr Val Ser Ala Leu Ile Pro Lys Pro Asn Val
130 135 140
Glu Gly Ile Tyr Leu Asp Glu Lys Ser Trp Asn Leu Glu Ser Ile Asp
145 150 155 160
Lys Ala Lys Thr Leu Pro Glu Ser Asp Pro Gln Lys Ser Leu Trp Val
165 170 175
Tyr Ala Ala Ser Lys Thr Glu Ala Glu Leu Ala Ala Trp Lys Phe Met
180 185 190
Asp Glu Asn Lys Pro His Phe Thr Leu Asn Ala Val Leu Pro Asn Tyr
195 200 205
Thr Ile Gly Thr Ile Phe Asp Pro Glu Thr Gln Ser Gly Ser Thr Ser
210 215 220
Gly Trp Met Met Ser Leu Phe Asn Gly Glu Val Ser Pro Ala Leu Ala
225 230 235 240
Leu Met Pro Pro Gln Tyr Tyr Val Ser Ala Val Asp Ile Gly Leu Leu
245 250 255
His Leu Gly Cys Leu Val Leu Pro Gln Ile Glu Arg Arg Arg Val Tyr
260 265 270
Gly Thr Ala Gly Thr Phe Asp Trp Asn Thr Val Leu Ala Thr Phe Arg
275 280 285
Lys Leu Tyr Pro Ser Lys Thr Phe Pro Ala Asp Phe Pro Asp Gln Gly
290 295 300
Gln Asp Leu Ser Lys Phe Asp Thr Ala Pro Ser Leu Glu Ile Leu Lys
305 310 315 320
Ser Leu Gly Arg Pro Gly Trp Arg Ser Ile Glu Glu Ser Ile Lys Asp
325 330 335
Leu Val Gly Ser Glu Thr Ala
340
 
<210>139
<211>1056
<212>DNA
<213> artificial sequence
 
<220>
<223> is from the codon optimized ADH-TB of the hot anerobe of Bu Shi
 
<400>139
atgaaaggct tcgccatgct gagcatcggc aaagtgggtt ggattgaaaa agaaaaaccg 60
gcgccaggcc cgttcgatgc aattgtgcgc cctctggcag tagcgccgtg taccagcgat 120
attcatactg tgtttgaagg tgccattggc gagcgtcaca atatgattct gggccatgaa 180
gccgttggtg aagttgttga ggttggcagc gaagtgaagg atttcaaacc gggcgatcgc 240
gttgtcgttc cagcgattac cccggattgg cgcaccagcg aagtccagcg cggctaccat 300
cagcactctg gcggcatgct ggccggctgg aaattcagca atgtaaagga tggtgtgttc 360
ggtgaatttt ttcacgttaa cgacgcagac atgaatctgg cgcacctgcc gaaagaaatc 420
ccgctggaag cagcggttat gattccggat atgatgacca cgggttttca cggcgcagag 480
ctggcggaca ttgaactggg cgctacggta gccgtactgg gcatcggtcc ggtgggcctg 540
atggcagttg caggcgctaa gctgcgcggc gcaggtcgta ttattgccgt tggttctcgc 600
ccggtgtgtg tggacgccgc taagtattat ggtgcaacgg acattgtcaa ttacaaggac 660
ggcccaattg aatctcagat catgaacctg acggaaggta aaggcgttga cgccgcgatt 720
atcgctggcg gcaacgccga catcatggcg accgcagtta aaatcgtcaa gccaggtggt 780
actattgcta acgtcaacta cttcggcgaa ggtgaggtcc tgcctgtccc acgtctggaa 840
tggggttgcg gtatggcaca taaaaccatt aaaggtggcc tgtgcccagg cggccgtctg 900
cgtatggaac gcctgatcga tctggtcttc tacaaacgcg tggatcctag caaactggtg 960
actcacgttt tccgcggctt tgataacatc gaaaaagctt ttatgctgat gaaagataaa 1020
ccgaaagatc tgattaaacc ggttgtcatc ctggct 1056
 
<210>140
<211>352
<212>PRT
<213> artificial sequence
 
<220>
<223> is from the ADH-TB of the hot anerobe of Bu Shi
 
<400>140
Met Lys Gly Phe Ala Met Leu Ser Ile Gly Lys Val Gly Trp Ile Glu
1 5 10 15
Lys Glu Lys Pro Ala Pro Gly Pro Phe Asp Ala Ile Val Arg Pro Leu
20 25 30
Ala Val Ala Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala
35 40 45
Ile Gly Glu Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu
50 55 60
Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg
65 70 75 80
Val Val Val Pro Ala Ile Thr Pro Asp Trp Arg Thr Ser Glu Val Gln
85 90 95
Arg Gly Tyr His Gln His Ser Gly Gly Met Leu Ala Gly Trp Lys Phe
100 105 110
Ser Asn Val Lys Asp Gly Val Phe Gly Glu Phe Phe His Val Asn Asp
115 120 125
Ala Asp Met Asn Leu Ala His Leu Pro Lys Glu Ile Pro Leu Glu Ala
130 135 140
Ala Val Met Ile Pro Asp Met Met Thr Thr Gly Phe His Gly Ala Glu
145 150 155 160
Leu Ala Asp Ile Glu Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly
165 170 175
Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Gly
180 185 190
Arg Ile Ile Ala Val Gly Ser Arg Pro Val Cys Val Asp Ala Ala Lys
195 200 205
Tyr Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asp Gly Pro Ile Glu
210 215 220
Ser Gln Ile Met Asn Leu Thr Glu Gly Lys Gly Val Asp Ala Ala Ile
225 230 235 240
Ile Ala Gly Gly Asn Ala Asp Ile Met Ala Thr Ala Val Lys Ile Val
245 250 255
Lys Pro Gly Gly Thr Ile Ala Asn Val Asn Tyr Phe Gly Glu Gly Glu
260 265 270
Val Leu Pro Val Pro Arg Leu Glu Trp Gly Cys Gly Met Ala His Lys
275 280 285
Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Arg
290 295 300
Leu Ile Asp Leu Val Phe Tyr Lys Arg Val Asp Pro Ser Lys Leu Val
305 310 315 320
Thr His Val Phe Arg Gly Phe Asp Asn Ile Glu Lys Ala Phe Met Leu
325 330 335
Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Ala
340 345 350
 
<210>141
<211>759
<212>DNA
<213> Lactobacillus minor
 
<400>141
atgaccgatc ggttgaaggg gaaagtagca attgtaactg gcggtacctt gggaattggc 60
ttggcaatcg ctgataagtt tgttgaagaa ggcgcaaagg ttgttattac cggccgtcac 120
gctgatgtag gtgaaaaagc tgccagatca atcggcggca cagacgttat ccgttttgtc 180
caacacgatg cttctgatga aaccggctgg actaagttgt ttgatacgac tgaagaagca 240
tttggcccag ttaccacggt tgtcaacaat gccggaattg cggtcagcaa gagtgttgaa 300
gataccacaa ctgaagaatg gcgcaagctg ctctcagtta acttggatgg tgtcttcttc 360
ggtacccgtc ttggaatcca acgtatgaag aataaaggac tcggagcatc aatcatcaat 420
atgtcatcta tcgaaggttt tgttggtgat ccagctctgg gtgcatacaa cgcttcaaaa 480
ggtgctgtca gaattatgtc taaatcagct gccttggatt gcgctttgaa ggactacgat 540
gttcgggtta acactgttca tccaggttat atcaagacac cattggttga cgatcttgaa 600
ggggcagaag aaatgatgtc acagcggacc aagacaccaa tgggtcatat cggtgaacct 660
aacgatatcg cttggatctg tgtttacctg gcatctgacg aatctaaatt tgccactggt 720
gcagaattcg ttgtcgacgg agggtacacc gcccaatag 759
 
<210>142
<211>252
<212>PRT
<213> Lactobacillus minor
 
<400>142
Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr
1 5 10 15
Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
35 40 45
Arg Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala
50 55 60
Ser Asp Glu Thr Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser
85 90 95
Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser
100 105 110
Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Glu Gly Phe Val Gly Asp Pro Ala Leu Gly Ala Tyr Asn Ala Ser Lys
145 150 155 160
Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
165 170 175
Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys
180 185 190
Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln
195 200 205
Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala
210 215 220
Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>143
<211>252
<212>PRT
<213> artificial sequence
 
<220>
<223> has the engineering ketoreductase list type of Lactobacillus minor skeleton
<223> synthesizes construct
 
<220> feature
<221>MISC_FEATURE
<222>(3)..(3)
<223>Xaa is polarity, acidity or aromatic moieties
 
<220> feature
<221>MISC_FEATURE
<222>(7)..(7)
<223>Xaa is polarity, nonpolar or restriction residue
 
<220> feature
<221>MISC_FEATURE
<222>(11)..(11)
<223>Xaa is aliphatics, nonpolar or polar residues
<220> feature
<221>MISC_FEATURE
<222>(16)..(16)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(19)..(19)
<223>Xaa is nonpolar or aliphatic residue
 
<220> feature
<221>MISC_FEATURE
<222>(23)..(23)
<223>Xaa is nonpolar or aromatic moieties
 
<220> feature
<221>MISC_FEATURE
<222>(41)..(41)
<223>Xaa is aliphatics, polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(45)..(45)
<223>Xaa is aliphatics, nonpolar or polar residues
 
<220> feature
<221>MISC_FEATURE
<222>(49)..(49)
<223>Xaa is alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(57)..(57)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(60)..(60)
<223>Xaa is aromatics, aliphatics, polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(64)..(64)
<223>Xaa is aliphatics or non-polar residue
<220> feature
<221>MISC_FEATURE
<222>(72)..(72)
<223>Xaa is alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(82)..(82)
<223>Xaa is polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(94)..(94)
<223>Xaa is polarity, nonpolar, alkaline or aliphatic residue
 
<220> feature
<221>MISC_FEATURE
<222>(95)..(95)
<223>Xaa is nonpolar or aliphatic residue
 
<220> feature
<221>MISC_FEATURE
<222>(96)..(96)
<223>Xaa is restriction, aliphatics, acidity, polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(97)..(97)
<223>Xaa is acid, alkaline or aliphatic residue
 
<220> feature
<221>MISC_FEATURE
<222>(106)..(106)
<223>Xaa is acidic residues
 
<220> feature
<221>MISC_FEATURE
<222>(108)..(108)
<223>Xaa is alkalescence, restriction or aromatic moieties
 
<220> feature
<221>MISC_FEATURE
<222>(111)..(111)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(117)..(117)
<223>Xaa is polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(126)..(126)
<223>Xaa be aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(127)..(127)
<223>Xaa is polarity or alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(145)..(145)
<223>Xaa is polar residues
 
<220> feature
<221>MISC_FEATURE
<222>(147)..(147)
<223>Xaa is aromatics, aliphatics, polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(152)..(152)
<223>Xaa is polarity, aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(157)..(157)
<223>Xaa is polarity or acidic residues
 
<220> feature
<221>MISC_FEATURE
<222>(163)..(163)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(173)..(173)
<223>Xaa is acid or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(177)..(177)
<223>Xaa is alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(192)..(192)
<223>Xaa is alkalescence or acidic residues
 
<220> feature
<221>MISC_FEATURE
<222>(194)..(194)
<223>Xaa is restriction, acidity, aliphatics, polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(198)..(198)
<223>Xaa is acid or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(200)..(200)
<223>Xaa is restriction, acid or alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(206)..(206)
<223>Xaa is polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(208)..(208)
<223>Xaa is polarity, restriction or alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(210)..(210)
<223>Xaa is aliphatics, polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(211)..(211)
<223>Xaa is alkalescence or acidic residues
 
<220> feature
<221>MISC_FEATURE
<222>(214)..(214)
<223>Xaa is nonpolar, aliphatics or polar residues
 
<220> feature
<221>MISC_FEATURE
<222>(217)..(217)
<223>Xaa is aromatics, aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(223)..(223)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(226)..(226)
<223>Xaa is nonpolar or aliphatic residue
 
<220> feature
<221>MISC_FEATURE
<222>(228)..(228)
<223>Xaa is nonpolar or aliphatic residue
 
<400>143
Met Thr Xaa Arg Leu Lys Xaa Lys Val Ala Xaa Val Thr Gly Gly Xaa
1 5 10 15
Leu Gly Xaa Gly Leu Ala Xaa Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Xaa Asp Val Gly Xaa Lys Ala Ala
35 40 45
Xaa Ser Ile Gly Gly Thr Asp Val Xaa Arg Phe Xaa Gln His Asp Xaa
50 55 60
Ser Asp Glu Thr Gly Trp Thr Xaa Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Xaa Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Xaa Xaa Xaa
85 90 95
Xaa Ser Val Glu Asp Thr Thr Thr Glu Xaa Trp Xaa Lys Leu Xaa Ser
100 105 110
Val Asn Leu Asp Xaa Val Phe Phe Gly Thr Arg Leu Gly Xaa Xaa Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Xaa Gly Xaa Val Gly Asp Pro Xaa Leu Gly Ala Tyr Xaa Ala Ser Lys
145 150 155 160
Gly Ala Xaa Arg Ile Met Ser Lys Ser Ala Ala Leu Xaa Cys Ala Leu
165 170 175
Xaa Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Xaa
180 185 190
Thr Xaa Leu Val Asp Xaa Leu Xaa Gly Ala Glu Glu Met Xaa Ser Xaa
195 200 205
Arg Xaa Xaa Thr Pro Xaa Gly His Xaa Gly Glu Pro Asn Asp Xaa Ala
210 215 220
Trp Xaa Cys Xaa Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>144
<211>252
<212>PRT
<213> artificial sequence
 
<220>
<223> has the engineering ketoreductase list type of short lactobacillus skeleton
<223> synthesizes construct
 
<220> feature
<221>MISC_FEATURE
<222>(3)..(3)
<223>Xaa is polarity, acidity or aromatic moieties
 
<220> feature
<221>MISC_FEATURE
<222>(7)..(7)
<223>Xaa is polarity, nonpolar or restriction residue
 
<220> feature
<221>MISC_FEATURE
<222>(11)..(11)
<223>Xaa is aliphatics, nonpolar or polar residues
<220> feature
<221>MISC_FEATURE
<222>(16)..(16)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(19)..(19)
<223>Xaa is nonpolar or aliphatic residue
 
<220> feature
<221>MISC_FEATURE
<222>(23)..(23)
<223>Xaa is nonpolar or aromatic moieties
 
<220> feature
<221>MISC_FEATURE
<222>(41)..(41)
<223>Xaa is aliphatics, polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(45)..(45)
<223>Xaa is aliphatics, nonpolar or polar residues
 
<220> feature
<221>MISC_FEATURE
<222>(49)..(49)
<223>Xaa is alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(57)..(57)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(60)..(60)
<223>Xaa is aromatics, aliphatics, polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(64)..(64)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(72)..(72)
<223>Xaa is alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(82)..(82)
<223>Xaa is polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(94)..(94)
<223>Xaa is polarity, nonpolar, alkaline or aliphatic residue
 
<220> feature
<221>MISC_FEATURE
<222>(95)..(95)
<223>Xaa is nonpolar or aliphatic residue
 
<220> feature
<221>MISC_FEATURE
<222>(96)..(96)
<223>Xaa is restriction, aliphatics, acidity, polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(97)..(97)
<223>Xaa is acid, alkaline or aliphatic residue
 
<220> feature
<221>MISC_FEATURE
<222>(106)..(106)
<223>Xaa is acidic residues
 
<220> feature
<221>MISC_FEATURE
<222>(108)..(108)
<223>Xaa is alkalescence, restriction or aromatic moieties
 
<220> feature
<221>MISC_FEATURE
<222>(111)..(111)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(117)..(117)
<223>Xaa is polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(126)..(126)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(127)..(127)
<223>Xaa is polarity or alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(145)..(145)
<223>Xaa is polar residues
 
<220> feature
<221>MISC_FEATURE
<222>(147)..(147)
<223>Xaa is aromatics, aliphatics, polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(152)..(152)
<223>Xaa is polarity, aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(157)..(157)
<223>Xaa is polarity or acidic residues
 
<220> feature
<221>MISC_FEATURE
<222>(163)..(163)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(173)..(173)
<223>Xaa is acid or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(177)..(177)
<223>Xaa is alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(192)..(192)
<223>Xaa is alkalescence or acidic residues
 
<220> feature
<221>MISC_FEATURE
<222>(194)..(194)
<223>Xaa is restriction, acidity, aliphatics, polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(198)..(198)
<223>Xaa is acid or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(200)..(200)
<223>Xaa is restriction, acid or alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(206)..(206)
<223>Xaa is polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(208)..(208)
<223>Xaa is polarity, restriction or alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(210)..(210)
<223>Xaa is aliphatics, polarity or non-polar residue
<220> feature
<221>MISC_FEATURE
<222>(211)..(211)
<223>Xaa is alkalescence or acidic residues
 
<220> feature
<221>MISC_FEATURE
<222>(214)..(214)
<223>Xaa is nonpolar, aliphatics or polar residues
 
<220> feature
<221>MISC_FEATURE
<222>(217)..(217)
<223>Xaa is aromatics, aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(223)..(223)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(226)..(226)
<223>Xaa is nonpolar or aliphatic residue
 
<220> feature
<221>MISC_FEATURE
<222>(228)..(228)
<223>Xaa is nonpolar or aliphatic residue
 
<400>144
Met Ser Xaa Arg Leu Asp Xaa Lys Val Ala Xaa Ile Thr Gly Gly Xaa
1 5 10 15
Leu Gly Xaa Gly Leu Ala Xaa Ala Thr Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Met Ile Thr Gly Arg His Xaa Asp Val Gly Xaa Lys Ala Ala
35 40 45
Xaa Ser Val Gly Thr Pro Asp Gln Xaa Gln Phe Xaa Gln His Asp Xaa
50 55 60
Ser Asp Glu Asp Gly Trp Thr Xaa Leu Phe Asp Ala Thr Glu Lys Ala
65 70 75 80
Phe Xaa Pro Val Ser Thr Leu Val Asn Asn Ala Gly Ile Xaa Xaa Xaa
85 90 95
Xaa Ser Val Glu Glu Thr Thr Thr Ala Xaa Trp Xaa Lys Leu Xaa Ala
100 105 110
Val Asn Leu Asp Xaa Val Phe Phe Gly Thr Arg Leu Gly Xaa Xaa Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Xaa Gly Xaa Val Gly Asp Pro Xaa Leu Gly Ala Tyr Xaa Ala Ser Lys
145 150 155 160
Gly Ala Xaa Arg Ile Met Ser Lys Ser Ala Ala Leu Xaa Cys Ala Leu
165 170 175
Xaa Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Xaa
180 185 190
Thr Xaa Leu Val Asp Xaa Leu Xaa Gly Ala Glu Glu Ala Xaa Ser Xaa
195 200 205
Arg Xaa Xaa Thr Pro Xaa Gly His Xaa Gly Glu Pro Asn Asp Xaa Ala
210 215 220
Tyr Xaa Cys Xaa Tyr Leu Ala Ser Asn Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ser Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>145
<211>252
<212>PRT
<213> artificial sequence
 
<220>
<223> has the engineering ketoreductase list type of lactobacillus kefir skeleton
<223> synthesizes construct
 
<220> feature
<221>MISC_FEATURE
<222>(3)..(3)
<223>Xaa is polarity, acidity or aromatic moieties
 
<220> feature
<221>MISC_FEATURE
<222>(7)..(7)
<223>Xaa is polarity, nonpolar or restriction residue
 
<220> feature
<221>MISC_FEATURE
<222>(11)..(11)
<223>Xaa is aliphatics, nonpolar or polar residues
<220> feature
<221>MISC_FEATURE
<222>(16)..(16)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(19)..(19)
<223>Xaa is nonpolar or aliphatic residue
 
<220> feature
<221>MISC_FEATURE
<222>(23)..(23)
<223>Xaa is nonpolar or aromatic moieties
 
<220>
<221>MISC_FEATURE
<222>(41)..(41)
<223>Xaa is aliphatics, polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(45)..(45)
<223>Xaa is aliphatics, nonpolar or polar residues
 
<220> feature
<221>MISC_FEATURE
<222>(49)..(49)
<223>Xaa is alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(57)..(57)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(60)..(60)
<223>Xaa is aromatics, aliphatics, polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(64)..(64)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(72)..(72)
<223>Xaa is alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(82)..(82)
<223>Xaa is polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(94)..(94)
<223>Xaa is polarity, nonpolar, alkaline or aliphatic residue
 
<220> feature
<221>MISC_FEATURE
<222>(95)..(95)
<223>Xaa is nonpolar or aliphatic residue
 
<220> feature
<221>MISC_FEATURE
<222>(96)..(96)
<223>Xaa is restriction, aliphatics, acidity, polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(97)..(97)
<223>Xaa is acid, alkaline or aliphatic residue
 
<220> feature
<221>MISC_FEATURE
<222>(106)..(106)
<223>Xaa is acidic residues
 
<220> feature
<221>MISC_FEATURE
 
<222>(108)..(108)
<223>Xaa is alkalescence, restriction or aromatic moieties
 
<220> feature
<221>MISC_FEATURE
<222>(111)..(111)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(117)..(117)
<223>Xaa is polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(126)..(126)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(127)..(127)
<223>Xaa is polarity or alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(145)..(145)
<223>Xaa is polar residues
 
<220> feature
<221>MISC_FEATURE
<222>(147)..(147)
<223>Xaa is aromatics, aliphatics, polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(152)..(152)
<223>Xaa is polarity, aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(157)..(157)
<223>Xaa is polarity or acidic residues
 
<220> feature
<221>MISC_FEATURE
<222>(163)..(163)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(173)..(173)
<223>Xaa is acid or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(177)..(177)
<223>Xaa is alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(192)..(192)
<223>Xaa is alkalescence or acidic residues
 
<220> feature
<221>MISC_FEATURE
<222>(194)..(194)
<223>Xaa is restriction, acidity, aliphatics, polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(198)..(198)
<223>Xaa is acid or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(200)..(200)
<223>Xaa is restriction, acid or alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(206)..(206)
<223>Xaa is polarity or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(208)..(208)
<223>Xaa is polarity, restriction or alkaline residue
 
<220> feature
<221>MISC_FEATURE
<222>(210)..(210)
<223>Xaa is aliphatics, polarity or non-polar residue
<220> feature
<221>MISC_FEATURE
<222>(211)..(211)
<223>Xaa is alkalescence or acidic residues
 
<220> feature
<221>MISC_FEATURE
<222>(214)..(214)
<223>Xaa is nonpolar, aliphatics or polar residues
 
<220> feature
<221>MISC_FEATURE
<222>(217)..(217)
<223>Xaa is aromatics, aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(223)..(223)
<223>Xaa is aliphatics or non-polar residue
 
<220> feature
<221>MISC_FEATURE
<222>(226)..(226)
<223>Xaa is nonpolar or aliphatic residue
 
<220> feature
<221>MISC_FEATURE
<222>(228)..(228)
<223>Xaa is nonpolar or aliphatic residue
 
<400>145
Met Thr Xaa Arg Leu Lys Xaa Lys Val Ala Xaa Val Thr Gly Gly Xaa
1 5 10 15
Leu Gly Xaa Gly Leu Ala Xaa Ala Asp Lys Phe Val Glu Glu Gly Ala
20 25 30
Lys Val Val Ile Thr Gly Arg His Xaa Asp Val Gly Xaa Lys Ala Ala
35 40 45
Xaa Ser Ile Gly Gly Thr Asp Val Xaa Arg Phe Xaa Gln His Asp Xaa
50 55 60
Ser Asp Glu Ala Gly Trp Thr Xaa Leu Phe Asp Thr Thr Glu Glu Ala
65 70 75 80
Phe Xaa Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Xaa Xaa Xaa
85 90 95
Xaa Ser Val Glu Asp Thr Thr Thr Glu Xaa Trp Xaa Lys Leu Xaa Ser
100 105 110
Val Asn Leu Asp Xaa Val Phe Phe Gly Thr Arg Leu Gly Xaa Xaa Arg
115 120 125
Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile
130 135 140
Xaa Gly Xaa Val Gly Asp Pro Xaa Leu Gly Ala Tyr Xaa Ala Ser Lys
145 150 155 160
Gly Ala Xaa Arg Ile Met Ser Lys Ser Ala Ala Leu Xaa Cys Ala Leu
165 170 175
Xaa Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Xaa
180 185 190
Thr Xaa Leu Val Asp Xaa Leu Xaa Gly Ala Glu Glu Met Xaa Ser Xaa
195 200 205
Arg Xaa Xaa Thr Pro Xaa Gly His Xaa Gly Glu Pro Asn Asp Xaa Ala
210 215 220
Trp Xaa Cys Xaa Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly
225 230 235 240
Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln
245 250
 
<210>146
<211>939
<212>DNA
<213> artificial sequence
 
<220>
<223> is from the YDL ADH of yeast saccharomyces cerevisiae
 
<400>146
atgtcttttc accaacagtt tttcacgctg aacaacggca ataaaatccc ggcgattgcc 60
atcatcggca ctggtacacg ttggtataaa aatgaagaaa ctgacgcgac cttctccaat 120
agtctggttg aacaaatcgt gtatgcgttg aaactgccgg ggattatcca catcgacgcc 180
gcggagattt atcgcaccta cccggaagtg ggtaaagcac tgtccctgac cgaaaagcct 240
cgtaacgcga tttttctgac ggataaatat tctccgcaga ttaaaatgag tgactcccct 300
gcggacggtc tggatttagc attgaagaaa atgggtacag attatgttga tttatatctg 360
ttacattccc cgtttgtttc gaaggaagtg aatggcttaa gcttagaaga ggcttggaaa 420
gatatggagc agttatacaa aagtggtaaa gctaaaaaca tcggggtttc caatttcgca 480
gtggaagacc tgcaacgtat cctgaaagtc gctgaagtta aacctcaggt caaccagatt 540
gagttctctc cgttcctgca aaaccaaaca ccaggcattt ataaattctg tcaggagcac 600
gatatcctgg tggaagcata ttctccgctg ggcccgctgc agaagaaaac cgcgcaggat 660
gacagccaac cattttttga gtacgtcaaa gaattgagcg aaaaatacat caaatccgag 720
gcccagatca tcctgcgctg ggtcactaaa cgcggtgtgc tgccagttac cacctcttca 780
aagcctcagc gcattagcga tgctcagaac ctgttttcct tcgacctgac agcggaagag 840
gttgataaaa tcacggagct gggtctggaa catgaaccgc tgcgcctgta ctggaataaa 900
ttgtatggca aatataacta cgccgcccag aaagtgtaa 939
 
<210>147
<211>312
<212>PRT
<213> artificial sequence
 
<220>
<223> is from the YDL ADH of yeast saccharomyces cerevisiae
 
<400>147
Met Ser Phe His Gln Gln Phe Phe Thr Leu Asn Asn Gly Asn Lys Ile
1 5 10 15
Pro Ala Ile Ala Ile Ile Gly Thr Gly Thr Arg Trp Tyr Lys Asn Glu
20 25 30
Glu Thr Asp Ala Thr Phe Ser Asn Ser Leu Val Glu Gln Ile Val Tyr
35 40 45
Ala Leu Lys Leu Pro Gly Ile Ile His Ile Asp Ala Ala Glu Ile Tyr
50 55 60
Arg Thr Tyr Pro Glu Val Gly Lys Ala Leu Ser Leu Thr Glu Lys Pro
65 70 75 80
Arg Asn Ala Ile Phe Leu Thr Asp Lys Tyr Ser Pro Gln Ile Lys Met
85 90 95
Ser Asp Ser Pro Ala Asp Gly Leu Asp Leu Ala Leu Lys Lys Met Gly
100 105 110
Thr Asp Tyr Val Asp Leu Tyr Leu Leu His Ser Pro Phe Val Ser Lys
115 120 125
Glu Val Asn Gly Leu Ser Leu Glu Glu Ala Trp Lys Asp Met Glu Gln
130 135 140
Leu Tyr Lys Ser Gly Lys Ala Lys Asn Ile Gly Val Ser Asn Phe Ala
145 150 155 160
Val Glu Asp Leu Gln Arg Ile Leu Lys Val Ala Glu Val Lys Pro Gln
165 170 175
Val Asn Gln Ile Glu Phe Ser Pro Phe Leu Gln Asn Gln Thr Pro Gly
180 185 190
Ile Tyr Lys Phe Cys Gln Glu His Asp Ile Leu Val Glu Ala Tyr Ser
195 200 205
Pro Leu Gly Pro Leu Gln Lys Lys Thr Ala Gln Asp Asp Ser Gln Pro
210 215 220
Phe Phe Glu Tyr Val Lys Glu Leu Ser Glu Lys Tyr Ile Lys Ser Glu
225 230 235 240
Ala Gln Ile Ile Leu Arg Trp Val Thr Lys Arg Gly Val Leu Pro Val
245 250 255
Thr Thr Ser Ser Lys Pro Gln Arg Ile Ser Asp Ala Gln Asn Leu Phe
260 265 270
Ser Phe Asp Leu Thr Ala Glu Glu Val Asp Lys Ile Thr Glu Leu Gly
275 280 285
Leu Glu His Glu Pro Leu Arg Leu Tyr Trp Asn Lys Leu Tyr Gly Lys
290 295 300
Tyr Asn Tyr Ala Ala Gln Lys Val
305 310
 
<210>148
<211>1047
<212>DNA
<213> artificial sequence
 
<220>
<223> is from the ADH of rhodococcus erythropolis
 
<400>148
atgaaagcca ttcagtacac tcgtatcggt gcggaaccag aactgactga aatcccgaag 60
ccggaaccgg gcccgggcga agtactgctg gaagtcacgg cagctggcgt gtgccattcc 120
gatgatttca ttatgtctct gccggaagaa cagtacacct acggcctgcc gctgaccctg 180
ggtcatgaag gtgctggtaa agttgccgca gttggcgaag gtgttgaagg gttggatatt 240
ggcaccaatg tggttgtgta cggcccatgg ggttgtggca actgttggca ttgcagtcag 300
ggcctggaga actattgctc ccgtgcgcag gaactgggta ttaacccgcc tggtctgggt 360
gctccggggg ctttggcaga atttatgatt gtcgactcac cacgtcattt ggtcccgatt 420
ggcgatttag accctgttaa aactgttccg ttgactgatg cgggcctgac cccataccat 480
gcaatcaaac gctccctgcc gaaactgcgc ggcggctctt atgcagtagt gatcggtacg 540
ggtggcctgg gccacgtggc tatccaactg ctgcgtcatt tatctgctgc aacggtgatc 600
gccttggacg tttctgccga taaactggaa ctggctacca aagtcggcgc acatgaagta 660
gtcctgtctg ataaagatgc agcggagaat gtgcgtaaaa ttactggtag ccaaggtgca 720
gctttggtgt tggattttgt gggctatcag cctaccattg acaccgccat ggcagtggcg 780
ggcgtgggct ctgacgtcac cattgttggt atcggtgatg gccaggcaca tgcgaaagtt 840
ggtttcttcc agagtcctta tgaggcatcg gttacggtac cttattgggg cgctcgtaat 900
gaactgatcg aattgatcga tctggcgcat gctggtattt tcgacattgc cgttgagacc 960
ttctctttgg ataatggtgc agaggcctat cgtcgcctgg ctgcgggcac actgtcaggc 1020
cgtgcggtag tcgtcccggg cctgtaa 1047
 
<210>149
<211>348
<212>PRT
<213> artificial sequence
<220>
<223> is from the ADH of rhodococcus erythropolis
 
<400>149
Met Lys Ala Ile Gln Tyr Thr Arg Ile Gly Ala Glu Pro Glu Leu Thr
1 5 10 15
Glu Ile Pro Lys Pro Glu Pro Gly Pro Gly Glu Val Leu Leu Glu Val
20 25 30
Thr Ala Ala Gly Val Cys His Ser Asp Asp Phe Ile Met Ser Leu Pro
35 40 45
Glu Glu Gln Tyr Thr Tyr Gly Leu Pro Leu Thr Leu Gly His Glu Gly
50 55 60
Ala Gly Lys Val Ala Ala Val Gly Glu Gly Val Glu Gly Leu Asp Ile
65 70 75 80
Gly Thr Asn Val Val Val Tyr Gly Pro Trp Gly Cys Gly Asn Cys Trp
85 90 95
His Cys Ser Gln Gly Leu Glu Asn Tyr Cys Ser Arg Ala Gln Glu Leu
100 105 110
Gly Ile Asn Pro Pro Gly Leu Gly Ala Pro Gly Ala Leu Ala Glu Phe
115 120 125
Met Ile Val Asp Ser Pro Arg His Leu Val Pro Ile Gly Asp Leu Asp
130 135 140
Pro Val Lys Thr Val Pro Leu Thr Asp Ala Gly Leu Thr Pro Tyr His
145 150 155 160
Ala Ile Lys Arg Ser Leu Pro Lys Leu Arg Gly Gly Ser Tyr Ala Val
165 170 175
Val Ile Gly Thr Gly Gly Leu Gly His Val Ala Ile Gln Leu Leu Arg
180 185 190
His Leu Ser Ala Ala Thr Val Ile Ala Leu Asp Val Ser Ala Asp Lys
195 200 205
Leu Glu Leu Ala Thr Lys Val Gly Ala His Glu Val Val Leu Ser Asp
210 215 220
Lys Asp Ala Ala Glu Asn Val Arg Lys Ile Thr Gly Ser Gln Gly Ala
225 230 235 240
Ala Leu Val Leu Asp Phe Val Gly Tyr Gln Pro Thr Ile Asp Thr Ala
245 250 255
Met Ala Val Ala Gly Val Gly Ser Asp Val Thr Ile Val Gly Ile Gly
260 265 270
Asp Gly Gln Ala His Ala Lys Val Gly Phe Phe Gln Ser Pro Tyr Glu
275 280 285
Ala Ser Val Thr Val Pro Tyr Trp Gly Ala Arg Asn Glu Leu Ile Glu
290 295 300
Leu Ile Asp Leu Ala His Ala Gly Ile Phe Asp Ile Ala Val Glu Thr
305 310 315 320
Phe Ser Leu Asp Asn Gly Ala Glu Ala Tyr Arg Arg Leu Ala Ala Gly
325 330 335
Thr Leu Ser Gly Arg Ala Val Val Val Pro Gly Leu
340 345
 
<210>150
<211>1047
<212>DNA
<213> artificial sequence
 
<220>
<223> is from the YGL ADH of yeast saccharomyces cerevisiae
 
<400>150
atgaccacgg aaaaaaccgt agtgttcgtg tcaggcgcga ccggttttat tgctctgcac 60
gtggtagatg acctgctgaa aactggttac aaagtaattg gttccggtcg ttctcaggaa 120
aaaaatgacg gtttgctgaa gaagttcaag tccaacccga atctgagcat ggaaattgtg 180
gaagatattg cggcaccaaa cgccttcgat aaagtattcc agaaacatgg taaagaaatt 240
aaagtggtcc tgcatatcgc gtccccggtc catttcaaca ctaccgattt cgaaaaagac 300
ttactgatcc cggcggtaaa cggtaccaaa tctattttgg aagcaattaa gaactatgcc 360
gcagacaccg tggaaaaagt ggttattact tcatctgttg ccgcgttggc ctctccgggt 420
gatatgaaag ataccagctt cgtggttaac gaagaatcct ggaataaaga cacctgggaa 480
tcgtgtcagg cgaatgctgt gtccgcttat tgcggttcta aaaaattcgc agagaaaacg 540
gcgtgggact tcttggaaga aaaccagagc agcattaaat ttactctgtc cacgattaac 600
ccaggcttcg tttttggtcc gcagctgttc gccgactcct tgcgcaatgg tattaactct 660
agcagtgcga ttattgcgaa cctggtgtcg tataaattag gggataactt ctacaattat 720
agcggcccgt ttatcgacgt ccgtgacgtt tccaaagctc atctgctggc atttgagaaa 780
cctgaatgcg ccggtcagcg cctgtttctg tgcgaggata tgttctgttc ccaggaagcc 840
ctggacattc tgaacgaaga atttccacag ctgaagggca agatcgcaac gggcgaacct 900
ggcagcggct cgaccttcct gactaaaaat tgttgcaaat gcgacaatcg taaaactaaa 960
aacttgctgg gcttccagtt caacaaattc cgcgactgca ttgtcgatac tgcgtcccag 1020
ttgctggaag tgcaaagcaa aagctaa 1047
 
<210>151
<21l>348
<212>PRT
<213> artificial sequence
 
<220>
<223> is from the YGL ADH of yeast saccharomyces cerevisiae
<400>151
Met Thr Thr Glu Lys Thr Val Val Phe Val Ser Gly Ala Thr Gly Phe
1 5 10 15
Ile Ala Leu His Val Val Asp Asp Leu Leu Lys Thr Gly Tyr Lys Val
20 25 30
Ile Gly Ser Gly Arg Ser Gln Glu Lys Asn Asp Gly Leu Leu Lys Lys
35 40 45
Phe Lys Ser Asn Pro Asn Leu Ser Met Glu Ile Val Glu Asp Ile Ala
50 55 60
Ala Pro Asn Ala Phe Asp Lys Val Phe Gln Lys His Gly Lys Glu Ile
65 70 75 80
Lys Val Val Leu His Ile Ala Ser Pro Val His Phe Asn Thr Thr Asp
85 90 95
Phe Glu Lys Asp Leu Leu Ile Pro Ala Val Asn Gly Thr Lys Ser Ile
100 105 110
Leu Glu Ala Ile Lys Asn Tyr Ala Ala Asp Thr Val Glu Lys Val Val
115 120 125
Ile Thr Ser Ser Val Ala Ala Leu Ala Ser Pro Gly Asp Met Lys Asp
130 135 140
Thr Ser Phe Val Val Asn Glu Glu Ser Trp Asn Lys Asp Thr Trp Glu
145 150 155 160
Ser Cys Gln Ala Asn Ala Val Ser Ala Tyr Cys Gly Ser Lys Lys Phe
165 170 175
Ala Glu Lys Thr Ala Trp Asp Phe Leu Glu Glu Asn Gln Ser Ser Ile
180 185 190
Lys Phe Thr Leu Ser Thr Ile Asn Pro Gly Phe Val Phe Gly Pro Gln
195 200 205
Leu Phe Ala Asp Ser Leu Arg Asn Gly Ile Asn Ser Ser Ser Ala Ile
210 215 220
Ile Ala Asn Leu Val Ser Tyr Lys Leu Gly Asp Asn Phe Tyr Asn Tyr
225 230 235 240
Ser Gly Pro Phe Ile Asp Val Arg Asp Val Ser Lys Ala His Leu Leu
245 250 255
Ala Phe Glu Lys Pro Glu Cys Ala Gly Gln Arg Leu Phe Leu Cys Glu
260 265 270
Asp Met Phe Cys Ser Gln Glu Ala Leu Asp Ile Leu Asn Glu Glu Phe
275 280 285
Pro Gln Leu Lys Gly Lys Ile Ala Thr Gly Glu Pro Gly Ser Gly Ser
290 295 300
Thr Phe Leu Thr Lys Asn Cys Cys Lys Cys Asp Asn Arg Lys Thr Lys
305 310 315 320
Asn Leu Leu Gly Phe Gln Phe Asn Lys Phe Arg Asp Cys Ile Val Asp
325 330 335
Thr Ala Ser Gln Leu Leu Glu Val Gln Ser Lys Ser
340 345
 
<210>152
<211>939
<212>DNA
<213> artificial sequence
 
<220>
<223> is from the YPR ADH of yeast saccharomyces cerevisiae
 
<400>152
atgccggcaa cgttaaaaaa cagcagtgct accttaaaat taaacacagg tgcgagcatt 60
cctgtcctgg ggttcggcac ctggcgctct gtcgataaca acggctatca tagtgtaatt 120
gcggcgctga aagcggggta ccgtcatatc gatgctgcgg ccatctatct gaatgaagaa 180
gaagtcggcc gtgcgatcaa ggactccggt gttcctcgtg aagaaatttt tattaccacc 240
aaactgtggg gcaccgaaca acgcgatcca gaagcagccc tgaacaaatc tctgaaacgt 300
ctgggtctgg actatgtgga cctgtatctg atgcactggc cggtccctct gaaaacagac 360
cgtgtaactg acggtaacgt cctgtgcatc ccgaccctgg aagatggcac cgtggacatc 420
gataccaaag agtggaattt tattaaaacc tgggaactga tgcaggaatt gccgaaaact 480
ggtaagacca aagccgtcgg tgtgtccaat ttttccatca acaatatcaa agaactgctg 540
gaatcgccaa ataacaaggt cgttccagca accaatcaga tcgagattca tccgttgctg 600
ccgcaggatg aattaatcgc cttttgtaaa gaaaaaggca ttgtggtcga agcatatagc 660
ccattcggct ccgctaacgc cccgctgctg aaagaacagg cgattatcga tatggccaaa 720
aagcacggcg tcgaaccggc gcaactgatt atcagctggt cgattcagcg cggttatgtg 780
gtattggcca agtccgtaaa tccggagcgt atcgtgtcga actttaagat ttttaccctg 840
ccagaggatg atttcaaaac catctctaac ctgagcaaag tgcacggtac caaacgtgtc 900
gttgacatga aatggggctc atttccgatt tttcaataa 939
 
<210>153
<211>312
<212>PRT
<213> artificial sequence
 
<220>
<223> is from the YPR ADH of yeast saccharomyces cerevisiae
 
<400>153
Met Pro Ala Thr Leu Lys Asn Ser Ser Ala Thr Leu Lys Leu Asn Thr
1 5 10 15
Gly Ala Ser Ile Pro Val Leu Gly Phe Gly Thr Trp Arg Ser Val Asp
20 25 30
Asn Asn Gly Tyr His Ser Val Ile Ala Ala Leu Lys Ala Gly Tyr Arg
35 40 45
His Ile Asp Ala Ala Ala Ile Tyr Leu Asn Glu Glu Glu Val Gly Arg
50 55 60
Ala Ile Lys Asp Ser Gly Val Pro Arg Glu Glu Ile Phe Ile Thr Thr
65 70 75 80
Lys Leu Trp Gly Thr Glu Gln Arg Asp Pro Glu Ala Ala Leu Asn Lys
85 90 95
Ser Leu Lys Arg Leu Gly Leu Asp Tyr Val Asp Leu Tyr Leu Met His
100 105 110
Trp Pro Val Pro Leu Lys Thr Asp Arg Val Thr Asp Gly Asn Val Leu
115 120 125
Cys Ile Pro Thr Leu Glu Asp Gly Thr Val Asp Ile Asp Thr Lys Glu
130 135 140
Trp Asn Phe Ile Lys Thr Trp Glu Leu Met Gln Glu Leu Pro Lys Thr
145 150 155 160
Gly Lys Thr Lys Ala Val Gly Val Ser Asn Phe Ser Ile Asn Asn Ile
165 170 175
Lys Glu Leu Leu Glu Ser Pro Asn Asn Lys Val Val Pro Ala Thr Asn
180 185 190
Gln Ile Glu Ile His Pro Leu Leu Pro Gln Asp Glu Leu Ile Ala Phe
195 200 205
Cys Lys Glu Lys Gly Ile Val Val Glu Ala Tyr Ser Pro Phe Gly Ser
210 215 220
Ala Asn Ala Pro Leu Leu Lys Glu Gln Ala Ile Ile Asp Met Ala Lys
225 230 235 240
Lys His Gly Val Glu Pro Ala Gln Leu Ile Ile Ser Trp Ser Ile Gln
245 250 255
Arg Gly Tyr Val Val Leu Ala Lys Ser Val Asn Pro Glu Arg Ile Val
260 265 270
Ser Asn Phe Lys Ile Phe Thr Leu Pro Glu Asp Asp Phe Lys Thr Ile
275 280 285
Ser Asn Leu Ser Lys Val His Gly Thr Lys Arg Val Val Asp Met Lys
290 295 300
Trp Gly Ser Phe Pro Ile Phe Gln
305 310
 
<210>154
<211>1029
<212>DNA
<213> artificial sequence
<220>
<223> is from the GRE ADH of yeast saccharomyces cerevisiae
 
<400>154
atgtctgtgt tcgtgtcagg cgcgaatggt tttattgctc agcacatcgt agatctgctg 60
ctgaaagaag attacaaagt aattggttcc gcacgttctc aggaaaaagc tgaaaatttg 120
accgaagcct tcggtaacaa cccgaaattt agcatggaag tggtgcctga tattagcaaa 180
ctggatgcct tcgatcatgt attccagaaa catggtaaag atattaaaat cgtcctgcat 240
accgcgtccc cgttttgttt cgatattacc gattccgaac gtgacttact gatcccggcg 300
gtaaacggtg tcaaaggtat tttgcacagt attaagaaat atgccgcaga cagcgtggaa 360
cgcgtggttc tgacttcatc ttacgccgcg gtatttgata tggcgaagga aaacgataag 420
agcctgacct tcaacgaaga atcctggaat ccggcgacct gggaatcgtg tcagagtgat 480
ccggtgaacg cttattgcgg ttctaaaaaa ttcgcagaga aagcagcgtg ggaattcttg 540
gaagaaaacc gtgatagcgt gaaatttgag ctgacagcgg tcaacccagt ttacgttttt 600
ggtccgcaga tgttcgataa agatgttaaa aaacacttga acaccagctg cgaactggtg 660
aactctctga tgcatctgag ccctgaagat aaaattccgg aactgtttgg cggttacatc 720
gacgtccgtg acgttgcgaa agctcatctg gttgcatttc agaaacgtga aacaatcggt 780
cagcgcctga tcgtgtcgga ggcacgtttc acgatgcagg acgttctgga cattctgaac 840
gaagactttc cagtactgaa gggcaatatc ccggtcggca agcctggcag cggcgccacc 900
cataatactc tgggcgccac cctggacaat aaaaaaagca aaaaattgct gggcttcaaa 960
ttccgtaatc tgaaagagac tattgacgat actgcgtccc agatcctgaa attcgaaggt 1020
cgcatttaa 1029
 
<210>155
<211>342
<212>PRT
<213> artificial sequence
 
<220>
<223> is from the GRE ADH of yeast saccharomyces cerevisiae
 
<400>155
Met Ser Val Phe Val Ser Gly Ala Asn Gly Phe Ile Ala Gln His Ile
1 5 10 15
Val Asp Leu Leu Leu Lys Glu Asp Tyr Lys Val Ile Gly Ser Ala Arg
20 25 30
Ser Gln Glu Lys Ala Glu Asn Leu Thr Glu Ala Phe Gly Asn Asn Pro
35 40 45
Lys Phe Ser Met Glu Val Val Pro Asp Ile Ser Lys Leu Asp Ala Phe
50 55 60
Asp His Val Phe Gln Lys His Gly Lys Asp Ile Lys Ile Val Leu His
65 70 75 80
Thr Ala Ser Pro Phe Cys Phe Asp Ile Thr Asp Ser Glu Arg Asp Leu
85 90 95
Leu Ile Pro Ala Val Asn Gly Val Lys Gly Ile Leu His Ser Ile Lys
100 105 110
Lys Tyr Ala Ala Asp Ser Val Glu Arg Val Val Leu Thr Ser Ser Tyr
115 120 125
Ala Ala Val Phe Asp Met Ala Lys Glu Asn Asp Lys Ser Leu Thr Phe
130 135 140
Asn Glu Glu Ser Trp Asn Pro Ala Thr Trp Glu Ser Cys Gln Ser Asp
145 150 155 160
Pro Val Asn Ala Tyr Cys Gly Ser Lys Lys Phe Ala Glu Lys Ala Ala
165 170 175
Trp Glu Phe Leu Glu Glu Asn Arg Asp Ser Val Lys Phe Glu Leu Thr
180 185 190
Ala Val Asn Pro Val Tyr Val Phe Gly Pro Gln Met Phe Asp Lys Asp
195 200 205
Val Lys Lys His Leu Asn Thr Ser Cys Glu Leu Val Asn Ser Leu Met
210 215 220
His Leu Ser Pro Glu Asp Lys Ile Pro Glu Leu Phe Gly Gly Tyr Ile
225 230 235 240
Asp Val Arg Asp Val Ala Lys Ala His Leu Val Ala Phe Gln Lys Arg
245 250 255
Glu Thr Ile Gly Gln Arg Leu Ile Val Ser Glu Ala Arg Phe Thr Met
260 265 270
Gln Asp Val Leu Asp Ile Leu Asn Glu Asp Phe Pro Val Leu Lys Gly
275 280 285
Asn Ile Pro Val Gly Lys Pro Gly Ser Gly Ala Thr His Asn Thr Leu
290 295 300
Gly Ala Thr Leu Asp Asn Lys Lys Ser Lys Lys Leu Leu Gly Phe Lys
305 310 315 320
Phe Arg Asn Leu Lys Glu Thr Ile Asp Asp Thr Ala Ser Gln Ile Leu
325 330 335
Lys Phe Glu Gly Arg Ile
340

Claims (18)

1. an engineering Ketoreductase polypeptides, 3-ketone group tetramethylene sulfide Stereoselective can be reduced into (R)-3-hydroxy tetrahydro thiophene by it, described engineering Ketoreductase polypeptides is by SEQ ID NO:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106, 108, 110, 112, 126, 128, the aminoacid sequence composition of 130 or 134.
2. Ketoreductase polypeptides as claimed in claim 1, wherein said stereoselectivity be at least 90% per-cent steric isomer excessive.
3. Ketoreductase polypeptides as claimed in claim 1, wherein said stereoselectivity be at least 98% per-cent steric isomer excessive.
4. Ketoreductase polypeptides as claimed in claim 3, described Ketoreductase polypeptides by corresponding SEQID NO:6,8,10,18,20,22,24,26,28,30,34,36,38,40,42,50,52,54,58,62,66,70,72,76,78, the aminoacid sequence of 80 or 134 forms.
5. Ketoreductase polypeptides as claimed in claim 1, 3-ketone group tetramethylene sulfide can be changed into (R)-3-hydroxy tetrahydro thiophene with the speed of the wild-type Ketoreductase polypeptides more than SEQ ID NO:4 by it, wherein said Ketoreductase polypeptides is by corresponding SEQ ID NO:6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 30, 32, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 64, 66, 68, 70, 74, 76, 78, 80, 86, 88, 90, 92, 104, 106, 110, 112, 126, the aminoacid sequence composition of 130 or 134.
6. Ketoreductase polypeptides as claimed in claim 1, 3-ketone group tetramethylene sulfide Stereoselective excessively with the per-cent steric isomer of at least 95% can be reduced into (R)-3-hydroxy tetrahydro thiophene by it, the wild-type Ketoreductase polypeptides of itself and SEQ ID NO:4 contrasts, there is activity and the thermostability of improvement, and by corresponding SEQ ID NO:6, 8, 12, 14, 22, 24, 26, 30, 32, 38, 42, 44, 46, 50, 52, 56, 58, 60, 64, 66, 68, 70, 74, 76, 78, 80, 82, 86, 88, 90, 92, 104, 106, 110, the aminoacid sequence composition of 112 or 134.
7. polynucleotide, the Ketoreductase polypeptides of its coding according to any one in claim 1 to 6.
8. polynucleotide as claimed in claim 7, it is selected from by the following group formed: SEQ ID NO:5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43,45,47,49,51,53,55,57,59,61,63,65,67,69,71,73,75,77,79,81,83,85,87,89,91,93,95,97,99,101,103,105,107,109,111,113,115,117,119,121,123,125,127,129,131 and 133.
9. an expression vector, it comprises polynucleotide as claimed in claim 7 or 8, and described polynucleotide are operably connected to and are suitable for instructing at least one control sequence expressed in host cell.
10. a host cell, it comprises expression vector according to claim 9.
11. 1 kinds for substrate 3-ketone group tetramethylene sulfide Stereoselective being reduced into the method for product (R)-3-hydroxy tetrahydro thiophene, it comprises and described 3-ketone group tetramethylene sulfide and the Ketoreductase polypeptides described in any one in claim 1 to 6 is being applicable to contacting under the reaction conditions described 3-ketone group tetramethylene sulfide being reduced into described (R)-3-hydroxy tetrahydro thiophene.
12. methods as claimed in claim 11, wherein 3-ketone group tetramethylene sulfide is excessively reduced into (R)-3-hydroxy tetrahydro thiophene by the steric isomer with at least 70%.
13. methods as claimed in claim 11, wherein 3-ketone group tetramethylene sulfide is reduced into (R)-3-hydroxy tetrahydro thiophene by so that at least 90% steric isomer is excessive.
14. methods as claimed in claim 11, wherein said 3-ketone group tetramethylene sulfide is excessively reduced into (R)-3-hydroxy tetrahydro thiophene by the steric isomer with at least 98%.
15. methods as claimed in claim 11, wherein said substrate is at least 100g/L, and the described substrate of at least 90% is converted to product being less than in 24 hours, wherein said Ketoreductase polypeptides is 0.8-1.0g/L, and is selected from by the following group formed: SEQ ID NO:6,8,10,12,14,18,20,22,24,26,30,32,38,40,42,44,46,48,50,52,54,56,58,60,64,66,68,70,74,76,78,80,82,86,88,90,92,104,106,110,112,126,130 and 134.
16. methods as claimed in claim 11, wherein said substrate is at least 100g/L, and the described substrate of at least 90% is converted to product being less than in 20 hours, wherein said Ketoreductase polypeptides is 0.8-1.0g/L, and be selected from by SEQ ID NO:26,44,68 and 104 groups formed.
17. method as claimed in claim 11, wherein said method is used for the antibiotic synthesis of structural formula (III):
18. 1 kinds of compositions, it comprises 3-ketone group tetramethylene sulfide and Ketoreductase polypeptides according to claim 1.
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Families Citing this family (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101528917B (en) 2006-10-02 2015-07-29 科德克希思公司 The Statins pure for the preparation of stereoisomerism and the composition of synthetic intermediate thereof and method
JP2010517574A (en) 2007-02-08 2010-05-27 コデクシス， インコーポレイテッド Ketreductase and uses thereof
US7977078B2 (en) 2007-08-24 2011-07-12 Codexis, Inc. Ketoreductase polypeptides for the production of (R)-3-hydroxythiolane
US8748143B2 (en) 2007-09-13 2014-06-10 Codexis, Inc. Ketoreductase polypeptides for the reduction of acetophenones
CN101889081B (en) * 2007-09-28 2014-06-18 科德克希思公司 Ketoreductase polypeptides and uses thereof
JP5646328B2 (en) * 2007-10-01 2014-12-24 コデクシス， インコーポレイテッド Ketreductase polypeptide for the production of azetidinone
WO2010025238A2 (en) 2008-08-27 2010-03-04 Codexis, Inc. Ketoreductase polypeptides for the production of a 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine
WO2010027710A2 (en) 2008-08-27 2010-03-11 Codexis, Inc. Ketoreductase polypeptides and uses thereof
US8288141B2 (en) 2008-08-27 2012-10-16 Codexis, Inc. Ketoreductase polypeptides for the production of 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine
CN102186972B (en) 2008-08-29 2014-08-20 科德克希思公司 Ketoreductase polypeptides for the stereoselective production of (4s)-3[(5s)-5(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one
ES2575560T3 (en) 2009-08-19 2016-06-29 Codexis, Inc. Cetorreductase polypeptides to prepare phenylephrine
SI2510089T1 (en) 2009-12-08 2015-12-31 Codexis, Inc. Synthesis of prazole compounds
US9040262B2 (en) 2010-05-04 2015-05-26 Codexis, Inc. Biocatalysts for ezetimibe synthesis
EP2649187B1 (en) 2010-12-08 2017-11-22 Codexis, Inc. Biocatalysts and methods for the synthesis of armodafinil
DK2697662T3 (en) 2011-04-13 2018-07-30 Codexis Inc BIOCATALYTIC PROCEDURE FOR PREPARING ESLICARBAZEPIN AND ANALOGUES THEREOF
HUE046089T2 (en) * 2011-11-18 2020-01-28 Codexis Inc Biocatalysts for the preparation of hydroxy substituted carbamates
HUE036947T2 (en) 2012-05-11 2018-08-28 Codexis Inc Engineered imine reductases and methods for the reductive amination of ketone and amine compounds
WO2014150633A1 (en) 2013-03-15 2014-09-25 Merck Sharp & Dohme Corp. Immobilized ketoreductases and process for making and using immobilized ketoreductase
CN103421854A (en) * 2013-07-30 2013-12-04 苏州汉酶生物技术有限公司 Biological preparation method of (S)-3-(dimethylamino)-1-(thiophene-2-radical)-1-propyl alcohol
EP3049973B1 (en) 2013-09-27 2018-08-08 Codexis, Inc. Automated screening of enzyme variants
EP3068793B1 (en) 2013-11-13 2021-02-17 Codexis, Inc. Engineered imine reductases and methods for the reductive amination of ketone and amine compounds
ES2765511T3 (en) 2013-11-15 2020-06-09 Hoffmann La Roche Processes for the preparation of pyrimidinylcyclopentane compounds
US10093905B2 (en) 2014-04-22 2018-10-09 C-Lecta Gmbh Ketoreductases
SG11201703258PA (en) 2014-11-25 2017-05-30 Codexis Inc Engineered imine reductases and methods for the reductive amination of ketone and amine compounds
EP3256577B1 (en) * 2015-02-10 2020-08-26 Codexis, Inc. Ketoreductase polypeptides for the synthesis of chiral compounds
CN107354179B (en) * 2016-05-09 2021-02-19 上海医药工业研究院 Method for preparing chiral compound containing hydroxyl
CN106497996B (en) * 2016-10-11 2021-01-12 凯莱英医药集团（天津）股份有限公司 Enzymatic preparation method of chiral alcohol
EP3652328A1 (en) 2017-07-14 2020-05-20 c-LEcta GmbH Ketoreductases
CN109576312B (en) * 2017-09-29 2021-08-24 尚科生物医药（上海）有限公司 Method for preparing (R) -2-chloro-1- (3-hydroxyphenyl) ethanol
US11162081B2 (en) * 2018-01-22 2021-11-02 Jilin Asymchem Laboratories Co., Ltd. Ketoreductase mutant and application thereof
GB201802383D0 (en) * 2018-02-14 2018-03-28 Givaudan Sa Process
CN112513025A (en) 2018-07-16 2021-03-16 诺华股份有限公司 Chemical process for the preparation of phenylpiperidinylindole derivatives
CN112930402A (en) * 2018-09-26 2021-06-08 卡拉马祖控股股份有限公司 Enzymatic production of modified hop products
US10961550B2 (en) 2018-09-26 2021-03-30 Kalamazoo Holdings, Inc. Enzymatic process for production of modified hop products
US11591625B2 (en) 2018-09-26 2023-02-28 Kalamazoo Holdings, Inc. Enzymatic process for production of modified hop products
KR102308173B1 (en) * 2018-09-28 2021-10-06 씨제이제일제당 주식회사 A Novel Fructose C4 epimerases and Preparation Method for producing Tagatose using the same
KR102072311B1 (en) * 2018-11-08 2020-01-31 배재대학교 산학협력단 Enterococcus faecium KA84 and method for production of ornithine using the same
CN109943543B (en) * 2019-03-28 2020-12-11 上海健康医学院 Alcohol dehydrogenase mutant and preparation method and application thereof
US11396666B2 (en) 2019-04-19 2022-07-26 Api Corporation Method for producing (1R,3R)-3-(trifluoromethyl)cyclohexan-1-ol and intermediate thereof
CN110218718A (en) * 2019-06-19 2019-09-10 南京趣酶生物科技有限公司 Immobilization Ketoreductase mutant and its preparing the application in Nebivolol chirality alcohol intermediate and the like
CN112458142B (en) * 2020-12-11 2023-05-12 上海合全药物研发有限公司 Method for preparing (1R, 2R) -2-hydroxycyclovaleronitrile by biocatalysis
TW202413637A (en) * 2022-07-15 2024-04-01 瑞士商赫孚孟拉羅股份公司 Mutant ketoreductase with increased ketoreductase activity as well as methods and uses involving the same

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002086126A2 (en) * 2001-04-20 2002-10-31 Juelich Enzyme Products Gmbh Enzymatic method for the enantioselective reduction of keto compounds
EP1323830A2 (en) * 2001-12-27 2003-07-02 DSM Fine Chemicals Austria Nfg GmbH & Co KG Process for the preparation of heterocyclic (R)- and (S)-cyanohydrines
WO2005033094A2 (en) * 2003-10-01 2005-04-14 Basf Aktiengesellschaft Methods for the production of 3-methylamino-1-(thiene-2-yl)-propane-1-ol

Family Cites Families (61)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4443373A (en) 1982-03-30 1984-04-17 Schering Corporation Process for the production of antibiotic penems
US4530793A (en) 1982-11-29 1985-07-23 Schering Corporation Processes for the production of penems
US4619924A (en) 1984-05-18 1986-10-28 Pfizer Inc. 2-alkylthiopenem derivatives
US4739047A (en) 1985-10-17 1988-04-19 Pfizer Inc. Intermediates for 2-(1-oxo-3-thiolanyl)-2-penem antibiotics
US4695626A (en) 1986-07-29 1987-09-22 Pfizer Inc. 1-aza-4,5-dithiabicyclo [4.2.0] oct-2-en-8-one-2 carboxylate esters
WO1988008845A1 (en) 1987-05-11 1988-11-17 Pfizer Inc. Diastereomeric 5r,6s-6-(1r-hydroxyethyl)-2-(cis-1-oxo-3-thiolanylthio)-2-penem-3-carboxylic acids
US5191077A (en) 1987-05-11 1993-03-02 Pfizer Inc. Diastereomeric 5R,6S-6-(1R-hydroxyethyl)-2-(cis-1-oxo-3-thiolanylthio)-2-penem-3-carboxylic acids
US4874877A (en) 1988-04-19 1989-10-17 Pfizer Inc. Process for optically active 3-(methane-sulfonyloxy)thiolane and analogs
US5538867A (en) 1988-09-13 1996-07-23 Elf Aquitaine Process for the electrochemical regeneration of pyridine cofactors
US4864046A (en) 1989-02-09 1989-09-05 Pfizer Inc. Process for optically active 3-thiolanyl sulfonate esters
US4940823A (en) 1989-07-12 1990-07-10 Pfizer Inc. Process for optically active 3-thiolanyl sulfonate esters
DE4014573C1 (en) 1990-05-07 1991-10-10 Forschungszentrum Juelich Gmbh, 5170 Juelich, De
JP2681057B2 (en) 1990-10-01 1997-11-19 高砂香料工業株式会社 2,2'-bis (diphenylphosphino) -5,5 ', 6,6', 7,7 ', 8,8'-octahydro-1,1'-binaphthyl and transition metal having this as a ligand Complex
US5225339A (en) 1992-02-26 1993-07-06 The Scripps Research Institute Lactobacillus kefir alcohol dehydrogenase
JP3155107B2 (en) 1993-01-12 2001-04-09 ダイセル化学工業株式会社 Method for producing optically active 4-halo-3-hydroxybutyrate
US5605793A (en) 1994-02-17 1997-02-25 Affymax Technologies N.V. Methods for in vitro recombination
US6335160B1 (en) 1995-02-17 2002-01-01 Maxygen, Inc. Methods and compositions for polypeptide engineering
US5837458A (en) 1994-02-17 1998-11-17 Maxygen, Inc. Methods and compositions for cellular and metabolic engineering
ATE206460T1 (en) 1994-06-03 2001-10-15 Novo Nordisk Biotech Inc PURIFIED MYCELIOPTHHORA LACCASES AND NUCLEIC ACIDS CODING THEREOF
WO1996000787A1 (en) 1994-06-30 1996-01-11 Novo Nordisk Biotech, Inc. Non-toxic, non-toxigenic, non-pathogenic fusarium expression system and promoters and terminators for use therein
US5491077A (en) 1994-07-20 1996-02-13 Merck & Co., Inc. Microbial method
US5427933A (en) 1994-07-20 1995-06-27 Merck & Co., Inc. Reduction of phenylalkyl ketones to the corresponding (S)-hydroxy derivatives using mucor hiemalis IFO 5834
JPH08336393A (en) 1995-04-13 1996-12-24 Mitsubishi Chem Corp Production of optically active gamma-substituted-beta-hydroxybutyric ester
FI104465B (en) 1995-06-14 2000-02-15 Valio Oy Protein hydrolyzates for the treatment and prevention of allergies and their preparation and use
DE19610984A1 (en) 1996-03-21 1997-09-25 Boehringer Mannheim Gmbh Alcohol dehydrogenase and its use for the enzymatic production of chiral hydroxy compounds
US5891685A (en) 1996-06-03 1999-04-06 Mitsubishi Chemical Corporation Method for producing ester of (S)-γ-halogenated-β-hydroxybutyric acid
DK1036198T3 (en) 1997-12-08 2013-01-02 California Inst Of Techn Method for Preparation of Polynucleotide and Polypeptide Sequences
DE19812004A1 (en) 1998-03-19 1999-09-30 Forschungszentrum Juelich Gmbh Dehydrogenases with improved NAD dependence, their production and use
US6495023B1 (en) 1998-07-09 2002-12-17 Michigan State University Electrochemical methods for generation of a biological proton motive force and pyridine nucleotide cofactor regeneration
DE19857302C2 (en) 1998-12-14 2000-10-26 Forschungszentrum Juelich Gmbh Process for the enantioselective reduction of 3,5-dioxocarboxylic acids, their salts and esters
JP4221100B2 (en) 1999-01-13 2009-02-12 エルピーダメモリ株式会社 Semiconductor device
JP2002537831A (en) 1999-03-11 2002-11-12 イーストマン ケミカル カンパニー Enzymatic reduction with hydrogen by metal-catalyzed cofactor regeneration
EP1194582A1 (en) 1999-07-09 2002-04-10 Forschungszentrum Jülich Gmbh Method of reducing keto-carboxylic acids and their esters
AU1551701A (en) 1999-12-03 2001-06-12 Kaneka Corporation Novel carbonyl reductase, gene thereof and method of using the same
EP1272967A2 (en) 2000-03-30 2003-01-08 Maxygen, Inc. In silico cross-over site selection
DE10037101A1 (en) 2000-07-27 2002-02-07 Degussa Recombinantly produced enzymes with improved NAD (H) acceptance
US7202070B2 (en) 2000-10-31 2007-04-10 Biocatalytics, Inc. Method for reductive amination of a ketone using a mutated enzyme
WO2002077258A1 (en) 2001-03-22 2002-10-03 Bristol-Myers Squibb Company Stereoselective reduction of substituted acetophenone
JP4213524B2 (en) 2003-04-17 2009-01-21 ダイセル化学工業株式会社 Novel carbonyl reductase, polynucleotide containing DNA encoding the enzyme, method for producing the same, and method for producing optically active alcohol using the same
AU2004266100A1 (en) 2003-08-11 2005-03-03 Codexis, Inc. Enzymatic processes for the production of 4-substituted 3-hydroxybutyric acid derivatives and vicinal cyano, hydroxy substituted carboxylic acid esters
BRPI0413492A (en) 2003-08-11 2006-10-17 Codexis Inc polypeptide, polynucleotide, isolated nucleic acid sequence, expression vector, host cell, method of making a gdh polypeptide, and, composition
EP1654356A1 (en) 2003-08-11 2006-05-10 Codexis, Inc. Improved halohydrin dehalogenases and related polynucleotides
US7629157B2 (en) 2003-08-11 2009-12-08 Codexis, Inc. Ketoreductase polypeptides and related polynucleotides
US20080050787A1 (en) 2003-12-02 2008-02-28 Hazuki Nagai Method for Manufacturing Optically Active Tetrahydrothiophene Derivative and Method for Crystallization of Optically Active Tetrahydrothiophene-3-Ol
AU2005201221A1 (en) 2004-03-31 2005-10-20 Daiso Co., Ltd Process for Preparing 3-Hydroxythiolane
US7393667B2 (en) 2005-05-31 2008-07-01 Bristol-Myers Squibb Company Stereoselective reduction process for the preparation of pyrrolotriazine compounds
WO2007010944A1 (en) 2005-07-20 2007-01-25 Kaneka Corporation Method for producing optically active 2-(n-substituted aminomethyl)-3-hydroxybutyric acid ester
AT502395B1 (en) 2005-07-27 2007-03-15 Iep Gmbh OXIDOREDUCETASES FOR THE STEREOSELECTIVE REDUCTION OF KETOVER BINDINGS
CN101528917B (en) 2006-10-02 2015-07-29 科德克希思公司 The Statins pure for the preparation of stereoisomerism and the composition of synthetic intermediate thereof and method
JP2010517574A (en) 2007-02-08 2010-05-27 コデクシス， インコーポレイテッド Ketreductase and uses thereof
US7977078B2 (en) 2007-08-24 2011-07-12 Codexis, Inc. Ketoreductase polypeptides for the production of (R)-3-hydroxythiolane
US8748143B2 (en) 2007-09-13 2014-06-10 Codexis, Inc. Ketoreductase polypeptides for the reduction of acetophenones
CN101889081B (en) 2007-09-28 2014-06-18 科德克希思公司 Ketoreductase polypeptides and uses thereof
JP5646328B2 (en) 2007-10-01 2014-12-24 コデクシス， インコーポレイテッド Ketreductase polypeptide for the production of azetidinone
WO2010025238A2 (en) * 2008-08-27 2010-03-04 Codexis, Inc. Ketoreductase polypeptides for the production of a 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine
US8288141B2 (en) * 2008-08-27 2012-10-16 Codexis, Inc. Ketoreductase polypeptides for the production of 3-aryl-3-hydroxypropanamine from a 3-aryl-3-ketopropanamine
WO2010027710A2 (en) 2008-08-27 2010-03-11 Codexis, Inc. Ketoreductase polypeptides and uses thereof
CN102186972B (en) 2008-08-29 2014-08-20 科德克希思公司 Ketoreductase polypeptides for the stereoselective production of (4s)-3[(5s)-5(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one
ES2575560T3 (en) 2009-08-19 2016-06-29 Codexis, Inc. Cetorreductase polypeptides to prepare phenylephrine
DK2697662T3 (en) 2011-04-13 2018-07-30 Codexis Inc BIOCATALYTIC PROCEDURE FOR PREPARING ESLICARBAZEPIN AND ANALOGUES THEREOF
HUE046089T2 (en) 2011-11-18 2020-01-28 Codexis Inc Biocatalysts for the preparation of hydroxy substituted carbamates

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002086126A2 (en) * 2001-04-20 2002-10-31 Juelich Enzyme Products Gmbh Enzymatic method for the enantioselective reduction of keto compounds
EP1323830A2 (en) * 2001-12-27 2003-07-02 DSM Fine Chemicals Austria Nfg GmbH & Co KG Process for the preparation of heterocyclic (R)- and (S)-cyanohydrines
WO2005033094A2 (en) * 2003-10-01 2005-04-14 Basf Aktiengesellschaft Methods for the production of 3-methylamino-1-(thiene-2-yl)-propane-1-ol

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